REVIEW ARTICLE Biosynthesis of D-arabinose in mycobacteria – a novel bacterial pathway with implications for antimycobacterial therapy Beata A Wolucka Laboratory of Mycobacterial Biochemistry, Institute of Public Health, Brussels, Belgium Keywords cell wall biosynthesis; D-ribose; ethambutol; Mycobacterium tuberculosis; mycolic acid; polyisoprenoid glycolipid; review Correspondence B A Wolucka, Laboratory of Mycobacterial Biochemistry, Institute of Public Health, 642 Engeland Street, B-1180 Brussels, Belgium Fax: +32 373 3282 Tel: +32 373 3100 E-mail: bwolucka@pasteur.be (Received February 2008, revised March 2008, accepted 12 March 2008) doi:10.1111/j.1742-4658.2008.06395.x Decaprenyl-phospho-arabinose (b-d-arabinofuranosyl-1-O-monophosphodecaprenol), the only known donor of d-arabinose in bacteria, and its precursor, decaprenyl-phospho-ribose (b-d-ribofuranosyl-1-O-monophosphodecaprenol), were first described in 1992 En route to d-arabinofuranose, the decaprenyl-phospho-ribose 2¢-epimerase converts decaprenyl-phospho-ribose to decaprenyl-phospho-arabinose, which is a substrate for arabinosyltransferases in the synthesis of the cell-wall arabinogalactan and lipoarabinomannan polysaccharides of mycobacteria The first step of the proposed decaprenylphospho-arabinose biosynthesis pathway in Mycobacterium tuberculosis and related actinobacteria is the formation of d-ribose 5-phosphate from sedoheptulose 7-phosphate, catalysed by the Rv1449 transketolase, and ⁄ or the isomerization of d-ribulose 5-phosphate, catalysed by the Rv2465 d-ribose 5-phosphate isomerase d-Ribose 5-phosphate is a substrate for the Rv1017 phosphoribosyl pyrophosphate synthetase which forms 5-phosphoribosyl 1-pyrophosphate (PRPP) The activated 5-phosphoribofuranosyl residue of PRPP is transferred by the Rv3806 5-phosphoribosyltransferase to decaprenyl phosphate, thus forming 5¢-phosphoribosyl-monophospho-decaprenol The dephosphorylation of 5¢-phosphoribosyl-monophospho-decaprenol to decaprenyl-phospho-ribose by the putative Rv3807 phospholipid phosphatase is the committed step of the pathway A subsequent 2¢-epimerization of decaprenyl-phospho-ribose by the heteromeric Rv3790 ⁄ Rv3791 2¢-epimerase leads to the formation of the decaprenyl-phospho-arabinose precursor for the synthesis of the cell-wall arabinans in Actinomycetales The mycobacterial 2¢-epimerase Rv3790 subunit is similar to the fungal d-arabinono-1,4-lactone oxidase, the last enzyme in the biosynthesis of d-erythroascorbic acid, thus pointing to an evolutionary link between the d-arabinofuranose- and l-ascorbic acidrelated pathways Decaprenyl-phospho-arabinose has been a lead compound for the chemical synthesis of substrates for mycobacterial arabinosyltransferases and of new inhibitors and potential antituberculosis drugs The peculiar (x,mono-E,octa-Z) configuration of decaprenol has yielded insights into lipid biosynthesis, and has led to the identification of the novel Z-polyprenyl diphosphate synthases of mycobacteria Mass spectrometric methods were developed for the analysis of anomeric linkages and of dolichol phosphaterelated lipids In the field of immunology, the renaissance in mycobacterial polyisoprenoid research has led to the identification of mimetic mannosyl-b1-phosphomycoketides of pathogenic mycobacteria as potent lipid antigens presented by CD1c proteins to human T cells Abbreviations ALO, D-arabinono-1,4-lactone oxidase; Araf, D-arabinofuranose; GLO, L-gulono-1,4-lactone oxidase; PRPP, 5-phosphoribosyl 1-pyrophosphate FEBS Journal 275 (2008) 2691–2711 ª 2008 The Author Journal compilation ª 2008 FEBS 2691 A role for the D-arabinose lipid carrier B A Wolucka The family of mycobacteria comprises about 100 species, several of which are pathogens of humans and ⁄ or animals, including Mycobacterium tuberculosis, M bovis, M leprae, M avium-intracellulare, M ulcerans and M marinum The pathogenic mycobacteria are inherently resistant to many antibacterial drugs and can persist for years inside infected cells Mycobacterium tuberculosis, the aetiological agent of tuberculosis, kills about 1.7 million people per year [1] and, according to World Health Organization estimations, is present in a latent form in about one-third of the world’s population (http://www.who.int/tb/en) A combination of several factors, such as the requirement of long-term multidrug therapy for the treatment of tuberculosis, the synergy between M tuberculosis and human immunodeficiency virus infections [2], the emergence of multidrug-resistant strains and, in particular, the recent outbreaks of extensively drug-resistant tuberculosis [3,4], has contributed to the persistence of tuberculosis as a global public health problem Several existing antituberculosis drugs, including the first-line drugs isoniazid and ethambutol, act at the level of the cell wall This vital structure plays a crucial role in the virulence and pathogenicity of M tuberculosis Mycobacteria possess a thick, highly impermeable hydrophobic cell wall composed of a thin layer of peptidoglycan, d-arabinofuranose (Araf)-containing arabinogalactan and arabinomannan polysaccharides, mannans, glucans, long-chain (C70–C90) a-branched, b-hydroxy fatty acids (mycolic acids) and other lipids, glycolipids, poly-l-glutamate–glutamine polymers, enzymes and other proteins Like teichoic acids in other Gram-positive bacteria [5], arabinogalactan is covalently attached to peptidoglycan by a phosphodiester linkage The arabinan part of arabinogalactan is, in turn, esterified to mycolic acids, thus forming a peptidoglycan–arabinogalactan–mycolic acid skeleton (reviewed in [6]) This rigid model of the mycobacterial cell wall is now being replaced by a more dynamic picture, in which the cell wall undergoes substantial modifications in response to changing growth conditions, as may occur in host cells, for example, after the proposed transfer from phagosomal to cytosolic compartments [7] The plasma membrane-anchored lipoarabinomannans and lipomannans, reminiscent of lipoteichoic acid, are probably translocated to the outer layer of the cell wall and processed to lipid-free arabinomannans and mannans [8] The presence, at least transient, of different proteins and enzymes in the M tuberculosis cell wall, such as the porins that are involved in the transport of hydrophilic molecules [9,10], the catalase-peroxidase katG [11,12], the heat shock protein 60 chaperones (GroEL1) that assist lipid 2692 traffic [13,14], the antigen 85 mycolyltransferases [15] complexed with a histone-like protein [16], the glutamine synthetase involved in the synthesis of poly-lglutamate–glutamine polymers [17], serine ⁄ threonine protein kinases [18–20] and other virulence factors [21,22], points to the dynamic structure, and suggests an active role of the organelle in host–pathogen interactions Indeed, profound alterations of the cell-wall composition are thought to occur that could lead to antigenic variation [23] and isoniazid resistance [24] of non-replicating, dormant M tuberculosis found in persistent infections Moreover, during human infection, the pathogen elaborates new macromolecular structures at the cell surface: pili, putative host colonization factors [25] d-Arabinose occurs rarely in nature In contrast with d-arabinopyranose, which is found in some eukaryotes, such as trypanosomatids and plants, Araf is confined to the prokaryotic world, where it is a constituent of cell-surface polymers and glycolipids In mycobacteria and related Actinomycetales species, Araf is a component of the arabinan parts of the arabinogalactan and (lipo)arabinomannan polymers of the cell wall and of some glycerol-based glycolipids [26] The branched arabinan chains of the arabinogalactan are attached to the linear galactan backbone The arabinan consists of an inner linear region of Araf-(1 fi 5)-a-Araf and of branched non-reducing terminal Ara6 motifs: Arafb1 fi 2Arafa1 fi 5(Arafb1 fi 2Arafa1 fi 3)Arafa1 fi 5Arafa1 About two-thirds of the terminal b-Araf and the penultimate 2-a-Araf serve as attachment sites for mycolic acids (reviewed in [6]) The arabinan part of the M tuberculosis lipoarabinomannan consists of linear segments of Araf(1 fi 5)-a-Araf with some a(1 fi 3) branching The non- reducing termini are composed of two distinct motifs: the Ara6 motif similar to that present in arabinogalactan, and a simplified linear Ara4 motif: Arafb fi 2Arafa1 fi 5Arafa1 fi 5Arafa1 Some of the non-reducing arabinofuranose termini are capped with short chains of a(1 fi 2) d-mannose [27] The physiological role of arabinans was thought to be exclusively structural and of similar importance within Corynebacterineae (the mycobacteria ⁄ nocardia ⁄ corynebacteria group); however, recent studies have challenged this simplistic view For example, arabinandevoid mutants of corynebacteria can be obtained [28,29], whereas abrogation of arabinan synthesis is lethal in mycobacteria In addition, the complex regulation [30] and functions [31] of arabinan-assembling Emb proteins suggest that this polymer could play a role in sensing mechanisms and possibly other processes, in particular in pathogenic mycobacteria FEBS Journal 275 (2008) 2691–2711 ª 2008 The Author Journal compilation ª 2008 FEBS A role for the D-arabinose lipid carrier B A Wolucka Despite the efforts of many research groups, the biosynthesis of d-arabinose in mycobacteria was an enigma for many years until the isolation of decaprenyl -phospho-arabinose and its decaprenyl-phospho-ribose precursor in 1990, and the proposal of the last step of d-arabinose synthesis catalysed by a 2¢-epimerase (Scheme 1) [32] The subsequent structural characterization of both the b-d-arabinofuranosyl-1monophosphodecaprenol (Fig 1B) [33] and the b-d-ribofuranosyl-1-monophosphodecaprenol (Fig 1C) [34] allowed the biological origins of bacterial Araf to be deciphered, and a new era in the study of cell-wall biosynthesis in mycobacteria to be started The discovery: decaprenyl-phosphoarabinose, decaprenyl-phospho-ribose and other endogenous lipid-linked sugars of mycobacteria In spite of several claims of the existence of activated nucleotide and 1-phosphate derivatives of d-arabinose [35–37], water-soluble activated forms of d-arabinose, Scheme The original scheme of biosynthesis of D-arabinofuranosyl residues of the cell-wall arabinogalactan and lipoarabinomannan in mycobacteria, including a feedback mechanism and possible sites of action of ethambutol, an antituberculosis drug [32] Two possible sites of ethambutol are indicated: 1, inhibition of arabinosyltransferase activity; 2, inhibition of certain step(s) in the biosynthesis of the acceptor X, where X may be a polyprenyl-pyrophosphoryl-oligosaccharide or a growing polymer chain Note that option 2, namely the inhibition of arabinan synthase activity (Emb), was demonstrated later by others (see text and Fig 2) Araf, D-arabinofuranose; Ribf, D-ribofuranose Fig Decaprenyl phosphate and decaprenyl-phospho-monosaccharides of mycobacteria (A) The mycobacterial lipid carrier C50-decaprenyl phosphate has a unique stereoconfiguration and contains only one trans (E)-isoprene residue at its x-end [33] (see Fig 3) (B) Decaprenyl-phospho-arabinose, the only known D-arabinose donor for the synthesis of the cell-wall arabinogalactan and lipoarabinomannan in mycobacteria [32,33] (C) Decaprenyl-phospho-ribose, the direct precursor of the b-D-arabinofuranosylmonophosphodecaprenol donor (B) and the major form of the naturally occurring decaprenyl-phospho-sugars of mycobacteria [32,34] (D) The mycobacterial decaprenylphospho-mannose, a minor component [107] FEBS Journal 275 (2008) 2691–2711 ª 2008 The Author Journal compilation ª 2008 FEBS 2693 A role for the D-arabinose lipid carrier B A Wolucka such as d-arabinose phosphates and d-arabinose nucleotides, have never been demonstrated in mycobacteria Exogenously added d-arabinose is catabolized by a spontaneous M smegmatis mutant via an inducible, fungal-like pathway [32,38,39] that converts an aldopentose into a ketopentose [40] (Fig 2) In the mycobacterial pathway, d-arabinose is reduced by a NADPH-dependent d-arabinose dehydrogenase to 2694 d-arabinitol, and the latter compound is oxidized to d-xylulose by a NAD-dependent d-arabinitol dehydrogenase d-Xylulose can then be phosphorylated to d-xylulose 5-phosphate and enter the pentose phosphate cycle [32,39] In contrast with mycobacteria, the majority of bacteria use either isomerase ⁄ kinase or oxidation pathways for the utilization of pentoses [41,42] Interestingly, the oxidation of d-arabinose to FEBS Journal 275 (2008) 2691–2711 ª 2008 The Author Journal compilation ª 2008 FEBS A role for the D-arabinose lipid carrier B A Wolucka d-arabinono-1,4-lactone does not occur in mycobacteria [32,39], but in fungi, where it has been believed, at least until recently [43], to be involved in the biosynthesis of d-erythroascorbic acid [44] After a fruitless search for water-soluble intermediates of d-arabinose, we looked for lipid-linked pyrophospho-oligosaccharides similar to the dolichol-linked oligosaccharides of archaebacteria [45] Indeed, gradient-eluted DEAE-cellulose fractions of organic extracts from M smegmatis contained lipid-linked galactoseoligosaccharides, but also large amounts of monocharged, acid-labile arabinose, ribose and mannose linked to phosphorylated isoprenoid lipids, although some mycolic acids could be detected as well Subsequent analysis of the monocharged glycolipids by fastatom bombardment mass spectrometry demonstrated the presence of decaprenyl-phospho-pentoses and decaprenyl phosphate ions at m ⁄ z 909 and m ⁄ z 777, respectively [32] This was the beginning of a fruitful search that has led to the identification of the d-arabinose pathway, and to a better understanding of cell-wall biosynthesis and of the mechanism of action of ethambutol in mycobacteria In particular, we discovered that ethambutol does not interfere with decaprenyl-phosphoarabinose synthesis, and that the site of action of the drug is downstream in the arabinan pathway [32] Accordingly, it was proposed that: (a) decaprenyl-phospho-arabinose is synthesized via a 2¢-epimerization of decaprenyl-phospho-ribose, and serves as the donor of d-arabinofuranosyl residues in the biosynthesis of the cell-wall arabinogalactan and (lipo)arabinomann; (b) ethambutol inhibits an arabinosyltransferase or an arabinan-forming enzyme, and this inhibition results in the accumulation of decaprenyl-phospho-arabinose in mycobacteria; (c) the synthesis of the decaprenyl-phospho-ribose precursor is controlled by a feedback mechanism (Scheme 1) [32] These conclusions have proven to be correct and have served as the basis for further research The details of the decaprenyl-phospho-arabinose structure, including the determination of the absolute configuration, anomeric linkage and ring form of the d-arabinosyl residue, were solved later using combined proton-NMR spectroscopy, gas chromatography and mass spectrometry (Fig 1B) [33] NMR analysis also allowed the determination of the particular structure of the mycobacterial decaprenol with important implications regarding its biosynthesis (Figs 1A and 3) It was a big surprise for us to find that what is lacking in the 10 isoprene unit-containing C50-decaprenol of mycobacteria is not a cis (Z)-unit, but one of the two trans (E)-isoprene units that are localized at the x-end of the known polyisoprenyl lipid carriers, including the common bacterial undecaprenol The proposed x,mono-E,octa-Z configuration of the mycobacterial decaprenol [33] was, in fact, the first hint of the existence of unusual Z-prenyl diphosphate synthases in mycobacteria: a Z-farnesyl diphosphate synthase that would provide an x,E,Z-farnesyl diphosphate for a subsequent specific enzyme, a Z-decaprenyl diphosphate synthase These unusual enzymes have been identified recently (see below) The structure of the endogenous b-d-arabinofuranosyl-1-monophosphodecaprenol of mycobacteria was solved (Fig 1B) This was unprecedented because, until that time, no other natural lipid-linked sugar isolated from an organism had been fully structurally characterized [46,47] The next step was the structural elucidation of decaprenyl-phospho-ribose (Fig 1C) [34] The presence of substantial amounts of decaprenyl-phospho-ribose was puzzling because no ribose-containing polymers have ever been described in mycobacteria We proposed that decaprenyl-phospho-ribose is converted to decaprenylphospho-d-arabinose by a novel 2¢-epimerase of mycobacteria (Scheme 1) [32] The decaprenylphospho-ribose 2¢-epimerase has been identified recently Fig The metabolism of D-arabinose in mycobacteria The fungal-like assimilation pathway for D-arabinose of Mycobacterium smegmatis [32,39] is shown (top reactions) Decaprenyl-phospho-D-arabinose, the only known D-arabinofuranose donor, and decaprenyl-phospho-ribose (in rectangles), were isolated from M smegmatis [32] and structurally characterized (see Fig 1) Decaprenyl-phospho-arabinose was proposed to be synthesized via a 2¢-epimerization of decaprenyl-phospho-ribose, and to control the synthesis of the latter compound by a feedback mechanism The heteromeric decaprenyl-phospho-ribose 2¢-epimerase (Rv3790 ⁄ Rv3791) was identified recently Ethambutol, a first-line drug for the treatment of tuberculosis, inhibits the utilization of decaprenyl-phospho-arabinose [32,33] at the level of the Emb proteins that are involved in the formation of arabinans [75,88] The enzymatic steps leading from the well-known 5-phosphoribosyl 1-pyrophosphate (PRPP) intermediate to the formation of decaprenyl-phospho-ribose were identified later by in vitro assays D-Ribose 5-phosphate, the direct precursor of PRPP, is proposed to be synthesized mainly by an essential transketolase (Rv1449) of the non-oxidative pentose phosphate pathway A possible involvement of a non-essential ribose 5-phosphate isomerase (Rv2465) and of the oxidative pentose phosphate pathway enzymes is also shown Intermediates of the fungal-like catabolic pathway are shown in green; the non-oxidative and oxidative parts of the pentose phosphate pathway are shown in blue and violet, respectively; the decaprenyl-phospho-arabinose pathway is shown in red Essential genes of M tuberculosis, as determined by Himar1-based transposon mutagenesis [52,133], are indicated in bold, and cloned genes are underlined FEBS Journal 275 (2008) 2691–2711 ª 2008 The Author Journal compilation ª 2008 FEBS 2695 A role for the D-arabinose lipid carrier B A Wolucka Fig The biosynthesis of C50-decaprenyl pyrophosphate in Mycobacterium tuberculosis The particular structure of the mycobacterial decaprenol (see Fig 1A) implies the existence in mycobacteria of unique Z-prenyl diphosphate synthases that use x,E-geranyl pyrophosphate as a substrate The nonessential Rv1086 Z-farnesyl diphosphate synthase and the essential Rv2361 Z-decaprenyl diphosphate synthase have been identified [71,72] The discovery of decaprenyl-phospho-ribose pointed to the involvement of activated ribose derivatives in the biosynthesis pathway to d-arabinose This observation was crucial for the identification of the precursor of decaprenyl-phospho-ribose An obvious candidate to test as a donor of the activated d-ribofuranosyl residue was the well-known, high-energy bond-containing 2696 intermediate for nucleotide synthesis: 5-phosphoribosyl 1-pyrophosphate (PRPP) In vitro assays of crude membranes of M smegmatis incubated with [14C]labelled PRPP and synthetic decaprenyl phosphate as substrates demonstrated the synthesis of decaprenylphospho-ribose 5¢-phosphate, which, on dephosphorylation, produces decaprenyl-phospho-ribose [37] The FEBS Journal 275 (2008) 2691–2711 ª 2008 The Author Journal compilation ª 2008 FEBS B A Wolucka A role for the D-arabinose lipid carrier progress of the mycobacterial genome sequencing projects [48,49] has allowed a comparative genomics approach that has led to the identification of the mycobacterial decaprenyl-phospho-ribose 2¢-epimerase and the phosphoribosyl transferase, involved in the biosynthesis of decaprenyl-phospho-arabinose [50] and decaprenyl-phospho-ribose [51], respectively The proposed pathway for the biosynthesis of b-D-arabinofuranosyl1-O-monophosphodecaprenol in mycobacteria Synthesis of D-ribose 5-phosphate The first step in the biosynthesis of the b-d-arabinofuranosyl-1-O-monophosphodecaprenol (decaprenylphospho-arabinose) in mycobacteria (Fig 2) is the synthesis of d-ribose 5-phosphate d-Ribose 5-phosphate could be synthesized by an amphibolic, thiamine diphosphate-dependent transketolase (vitamin B1) (sedoheptulose 7-phosphate:d-glyceraldehyde 3-phosphate glycolaldehydetransferase; EC 2.2.1.1), which reversibly transfers a keto group from sedoheptulose 7-phosphate to d-glyceraldehyde 3-phosphate, and produces d-ribose 5-phosphate and d-xylulose 5-phosphate, according to reaction (1): sedoheptulose 7-phosphateỵ D-glyceraldehyde 3-phosphate , D -ribose 5-phosphateỵ D-xylulose5-phosphate 1ị The transketolase is a ubiquitous enzyme that links the glycolytic and pentose phosphate pathways, but has never been studied in mycobacteria The M tuberculosis genome contains one sequence encoding a putative transketolase (Rv1449), and the gene is essential [52] Otherwise, d-ribose 5-phosphate could be formed from another intermediate of the pentose phosphate pathway, d-ribulose 5-phosphate, by the ribose 5-phosphate isomerase (Rv2465) (Fig 2) Surprisingly, the ribose 5-phosphate isomerase, and also several other pentose phosphate pathway genes, such as d-xylulose 5-phosphate 3-epimerase (Rv1408) and the 6-phosphoglucono-1,5-lactone lactonase (Rv1445), are apparently not essential in M tuberculosis [52] Consequently, the reaction catalysed by the ribose 5-phosphate isomerase probably plays a minor role in the synthesis of the vital arabinans in mycobacteria Formation of 5-phosphoribosyl-a-1-pyrophosphate The second step in the biosynthesis of decaprenylphospho-arabinose (Fig 2) is the reaction of ribose 5-phosphate with ATP to yield 5-phosphoribosyl-a-1pyrophosphate and AMP, catalysed by a PRPP synthetase (ribose 5-phosphate diphosphokinase; EC 2.7.6.1) (reaction 2): ribose 5-phosphate ỵ ATP , 5-phospho-a-D -ribose 1-pyrophosphate ỵ AMP 2ị PRPP is a key metabolite in the purine and pyrimidine nucleotide de novo and salvage pathways, the biosynthesis of pyridine nucleotide coenzymes and the synthesis of histidine and tryptophan By analogy with the decaprenyl-phospho-arabinose biosynthesis of mycobacteria, PRPP is proposed to be a precursor of b-dribofuranosyl residues of lipopolysaccharides and capsular polysaccharides of Gram-negative bacteria, such as Pseudomonas aeruginosa, Salmonella sp., Shigella sp., Escherichia coli, Proteus sp., Haemophilus influenzae and, perhaps, of eukaryotic trypanosomatids [34] Mycobacterium tuberculosis contains one PRPP synthetase protein (Rv1017) that shares at least 43% identity with its human, plant and bacterial homologues The mycobacterial PRPP synthetase sequence contains a conserved PRK03092 domain from Val227 to Ala240 (VLIDDMIDTGGTIA) that corresponds to the PRPP binding motif The PRPP synthetases are known to undergo a complex regulation, and both ADP and inorganic phosphate (Pi) are the known allosteric regulators of the enzyme [53] In spite of its central role in cell-wall, nucleic acid and protein biosynthesis, the mycobacterial PRPP synthetase has not yet been characterized In Fig 2, we propose that the inhibition of arabinan synthesis by ethambutol, and the resulting accumulation of decaprenyl-phospho-arabinose [32,33], could have further repercussions via a feedback mechanism, and inhibit, directly or indirectly, the PRPP synthetase activity in mycobacteria This would result in decreased amounts of the PRPP precursor and, in agreement with the observed complex effects of the drug, lead to the inhibition of the synthesis of decaprenyl-phospho-ribose [32,33], but also of nucleic acids and other compounds [54] Synthesis of b-D-5¢-phosphoribosyl-1-monophosphodecaprenol The next step of the decaprenyl-phospho-arabinose pathway (Fig 2) is the reversible transfer of the 5-phosphoribosyl residue from the activated PRPP donor to the decaprenyl phosphate acceptor, catalysed by a 5-phospho-a-d-ribose 1-pyrophosphate:decaprenyl phosphate 5-phosphoribosyltransferase (reaction 3): FEBS Journal 275 (2008) 2691–2711 ª 2008 The Author Journal compilation ª 2008 FEBS 2697 A role for the D-arabinose lipid carrier B A Wolucka 5-phospho-a-D-ribose 1-pyrophosphate ỵdecaprenyl phosphate , 50 -phosphoribosyl -b-1-monophospho-decaprenol ỵ PPi 3ị On the basis of the determined chemical structure of b-d-ribosyl-1-monophosphodecaprenol [34], it can be predicted that the product of the ribosyltransferase reaction is b-d-5¢-phosphoribosyl-1-monophosphodecaprenol Thus, the reaction would occur with an inversion of the anomeric configuration of the 5-phosphoribosyl residue, although direct evidence is lacking The decaprenyl phosphate-dependent phosphoribosyltransferase activity was demonstrated in vitro using crude membranes from M smegmatis and a [14C]labelled PRPP substrate [37] It was claimed that polyprenylphosphate-5-phosphoarabinose was one of the reaction products and the direct precursor of polyprenyl-phospho-arabinose in mycobacteria, and it was concluded that the epimerization at the C2 position of the ribosyl residue takes place at the level of either phosphoribose pyrophosphate or polyprenylphosphate5-phosphoribose [36,37] The M tuberculosis genes encoding 5-phospho-a-dribose 1-pyrophosphate:decaprenyl phosphate 5-phosphoribosyltransferase (Rv3806) and the downstream enzyme decaprenyl-phospho-ribose 2¢-epimerase (Rv3790 ⁄ Rv3791) were identified only recently using a comparative genomics strategy, as suggested earlier [34], namely by searching M tuberculosis orthologues of the Azorhizobium genes that are involved in the d-arabinosylation of nodulation factor glycolipids [50,51] It is worth noting that the sequences of the Nod-factor genes for d-arabinosylation have never been published, and the gene functions are, in fact, unknown [51] In addition, the early work reported the presence of d-arabinose in the Azorhizobium Nod factor glycolipids in the pyranose rather than furanose form [55], and convincing evidence for the presence of Araf is lacking In contrast, the advent of the M tuberculosis and M leprae genome data [48,49] has played an indisputable role in the identification of genes for the mycobacterial arabinogalactan ⁄ arabinomannan synthesis, and led to the proposed d-arabinose pathway in mycobacteria Homologues of the Rv3806 protein (annotated as UbiA prenyltransferases) are present in some Archaea and in many eubacteria, such as mycobacteria, corynebacteria and nocardia that share a similar composition of their cell walls, certain species of cyanobacteria, gamma-proteobacteria, clostridia and others The Rv3806 phosphoribosyltransferase (302 amino acids) is an integral membrane protein that requires Mg2+ for its activity The unpurified recombinant enzyme pres2698 ent in the membrane of the E coli host had apparent Km values for PRPP and the decaprenyl phosphate of plant origin substrates of 120 and 22 lm, respectively [51] The enzyme had a preference for medium-chain polyprenyl phosphates (C50–C55) and showed no activity with a short-chain C20-polyprenyl phosphate The pH optimum for the phosphoribosyltransferase reaction was pH 7.5–8 Contrary to the authors’ claim [51], the reaction catalysed by the 5-phospho-a-dribose 1-pyrophosphate:decaprenyl phosphate 5-phosphoribosyltransferase is probably not the committed step of decaprenyl-phospho-arabinose biosynthesis, because it is reversible in the absence of pyrophosphatase activity Synthesis of b-D-ribosyl-1-monophosphodecaprenol (decaprenyl-phospho-ribose) Decaprenyl-phospho-ribose is the major form of the lipid-linked pentoses in mycobacteria [34] (Fig 1C) It is formed by the removal of a 5¢-phosphate group of the b-d-5¢-phosphoribosyl-1-monophosphodecaprenol precursor, catalysed by a phosphatase (reaction 4): 50 -phosphoribosyl-b-1-monophospho-decaprenol ! b-D-ribosyl-1-monophospho-decaprenol ỵ Pi 4ị The phosphatase reaction is expected to be irreversible, and thus it would represent the committed step in the biosynthesis of decaprenyl-phospho-arabinose in mycobacteria Inspection of the M tuberculosis operons involved in the biosynthesis of the arabinan and galactan polymers has revealed the presence of an unknown PAP2-family phospholipid phosphatase (Rv3807), which is located next to the phosphoribosyltransferase (Rv3806) discussed above The Rv3807 orthologues are present in all Corynebacterineae The Rv3807 protein is therefore a good candidate for a specific decaprenylphospho-ribose-5¢-phosphate phosphatase Surprisingly, the Rv3807 gene is apparently not essential [52], whereas all the other genes related to decaprenyl-phospho-arabinose synthesis are annotated as essential genes Further studies are necessary to elucidate the biological function of the Rv3807 gene product 2¢-Epimerization of decaprenyl-phospho-ribose to decaprenyl-phospho-arabinose The last step of the biosynthetic pathway of decaprenyl-phospho-arabinose (b-d-arabinofuranosyl-1-monophosphodecaprenol) is the 2¢-epimerization of d-ribofuranosyl to d-arabinofuranosyl at the level of decaprenyl-phospho-pentoses, as originally proposed FEBS Journal 275 (2008) 2691–2711 ª 2008 The Author Journal compilation ª 2008 FEBS B A Wolucka [32,33] (Fig 2) This conversion proceeds via a decaprenyl-phospho-2¢-keto-d-arabinose intermediate, which is probably not released from the mycobacterial enzyme under physiological conditions (reaction 5): b-D-ribofuranosyl-1-monophosphodecaprenol ! ẵ20 -keto-b-D-arabinofuranosyl1-monophosphodecaprenol ! b-D-arabinofuranosyl1-monophosphodecaprenol 5ị The decaprenyl-phospho-ribose 2Â-epimerase is a heteromeric enzyme composed of two types of polypeptide that are annotated as an oxidoreductase and a short-chain dehydrogenase ⁄ reductase, and encoded by the Rv3790 and Rv3791 genes, respectively, of the M tuberculosis genome [50] The exact composition of the enzyme is unknown However, simultaneous expression of both polypeptides is required for epimerase activity Close homologues of the Rv3790 and Rv3791 proteins are present in arabinan-synthesizing mycobacteria, corynebacteria, nocardia and related actinobacteria, but also in other bacteria, many of which are pathogens and symbionts of animals and plants: for example, Pseudomonas aeruginosa, Burkholderia sp., Legionella pneumophila, Leptospira interrogans and Rhizobium etli Interestingly, species that are known to contain Araf as a component of their lipopolysaccharide, such as the opportunistic pathogen Pseudomonas aeruginosa and the legume symbiont Sinorhizobium meliloti, possess sequences that are similar (35% identity) to the Rv3790 and Rv3791 subunits of the heteromeric 2¢-epimerase of M tuberculosis The Rv3790 oxidoreductase protein (461 amino acids) contains a FAD-binding N-terminal domain and a C-terminal d-arabinono-1,4-lactone oxidase (ALO) signature from T423 to L458 The ALO domain is characteristic for l-gulono-1,4-lactone oxidase (GLO)like enzymes that catalyse the last step in the biosynthesis of l-ascorbic acid (or its 5-carbon homologue d-erythroascorbic acid) in plants, animals, fungi and some microbes [56,57] The Rv3790 protein shares 22% identical residues with d-arabinono-1,4-lactone oxidase of Sacccharomyces cerevisiae (ALO1) [58] The protein also shows a limited identity at both the N- and C-termini (26% and 38% identity, respectively) with the recently identified l-gulono-1,4-lactone dehydrogenase (Rv1771) of M tuberculosis [59] The yeast ALO1 enzyme catalyses the last step of oxidation of d-arabinono-1,4-lactone to d-erythroascorbic acid, and uses molecular oxygen as electron acceptor The Rv1771 dehydrogenase is probably involved in the synthesis of l-ascorbic acid (vitamin C) in M tuberculosis; the enzyme is specific for l-gulono-1,4-lactone, and A role for the D-arabinose lipid carrier can use both cytochrome c and a phenazine derivative as electron acceptors [59] The d-arabinono-1,4-lactone substrate of the yeast ALO1 enzyme has a furan-based ring structure that is similar to the d-arabinofuranosyl moiety of the epimerase reaction product (Fig 5) Although the mechanism of GLO and other GLO-like enzymes is not well understood, the GLO-catalysed reaction is thought to proceed via oxidation of the 2-hydroxyl group to a 2-keto derivative, which subsequently undergoes an enolization to form l-ascorbic acid (or d-erythroascorbic acid) It is probable therefore that the Rv3790 subunit(s) is directly responsible for the conversion of decaprenyl-phospho-ribose to the corresponding 2¢-keto-b-d-erythropentofuranose derivative (Figs and 5) In conclusion, little is known about the Rv3790 ⁄ Rv3791 decaprenyl-phospho-ribose 2¢-epimerase of M tuberculosis In particular, the nature of the flavin cofactor of the Rv3790 subunit and of the electron acceptors has not been elucidated As discussed above, an evolutionary link exists between Araf and l-ascorbic acid ⁄ d-erythroascorbic acid biosynthesis pathways An ancestor GLO-like gene of Actinomycetales or an unrelated gene that has acquired an ALO-like domain by convergent evolution could evolve into an Araf synthesizing enzyme (Rv3790) by recruiting an ancient short-chain dehydrogenase ⁄ reductase (Rv3791) that reduces the 2¢-keto d-arabinofuranose ring to a d-arabinofuranosyl residue Interestingly, pathogenic actinobacteria, including M tuberculosis, M bovis, M ulcerans and M marinum, have acquired, via gene duplication ⁄ divergent evolution or horizontal gene transfer, another GLO gene (Rv1771 in M tuberculosis) for the synthesis of l-ascorbic acid (or a related compound) The product of the Rv1771-catalysed reaction might interfere with l-ascorbic acid-dependent signal transduction pathways of animal hosts; however, its functions in M tuberculosis are still unknown [59] Other activated forms of D-arabinose Decaprenyl-phospho-arabinose (b-d-arabinofuranosyl1-monophosphodecaprenol) is the only known donor of d-arabinofuranosyl units in the synthesis of arabinans of Actinomycetales Disruption of the gene encoding the 5-phospho-a-d-ribose 1-pyrophosphate:decaprenyl phosphate 5-phosphoribosyltransferase (UbiA) produces a d-arabinose-deficient mutant of Corynebacterium glutamicum that is devoid of the cellwall arabinan–corynomycolic acid complex [28] This surprising result indicates that both the arabinan part FEBS Journal 275 (2008) 2691–2711 ª 2008 The Author Journal compilation ª 2008 FEBS 2699 A role for the D-arabinose lipid carrier B A Wolucka and the bound corynomycolic acids of the cell-wall peptidoglycan–arabinogalactan–corynomycolate core are not essential for the survival of C glutamicum In contrast, the arabinan part of the peptidoglycan–arabinogalactan–mycolate core is essential in mycobacteria, because disruption of the priming arabinosyltransferase AftA (Rv3792), which adds the first d-arabinofuranosyl residue to the galactan core, or of the Rv3806 phosphoribosyl transferase, is lethal in M tuberculosis [28,60] Mycobacterium smegmatis synthesizes an additional compound containing an activated d-arabinose residue, namely a partially saturated b-d-arabinosyl-1monophospho-octahydroheptaprenol (Fig 4B) [61] The biosynthesis of the C35-isoprenyl-phospho derivative of d-arabinose is unknown, although it is possible that the compound is synthesized via the decaprenylphospho-arabinose pathway because of the low specificity of the decaprenyl-phosphate-dependent enzymes Otherwise, the C35-octahydroheptaprenyl-phosphoarabinose could be synthesized by a direct transfer of the d-arabinofuranosyl unit from decaprenyl-phosphoarabinose or another donor to the C35-octahydroheptaprenyl phosphate acceptor In agreement with the latter proposal, ribosylated derivatives of C35-octahydroheptaprenyl phosphate have never been described The biological function of b-d-arabinosyl-1-monophospho-octahydroheptaprenol of M smegmatis is unknown Moreover, it is not clear whether other mycobacteria synthesize C35-octahydroheptaprenylphosphate derivatives Interestingly, single terminal d-arabinofuranosyl residues of short lipoarabinomannans of C glutamicum are apparently not derived from decaprenyl-phospho-arabinose, but rather from another, still unknown, donor [62] In contrast with Araf, which is present exclusively in bacteria, d-arabinopyranose is found in polysaccharides Fig The partially and fully saturated glycosylated phospholipids of mycobacteria (A) The major form of the lipid-linked mannose in Mycobacterium smegmatis, the partially saturated short-chain C35-octahydroheptaprenyl-phospho-mannose [107] (B) A minor form of the lipid-linked D-arabinose of M smegmatis, the partially saturated shortchain C35-octahydroheptaprenyl-phosphoarabinose [61] (C) The mycolylated isoprenoid phospholipid of M smegmatis [120] (D) The C30-mannosyl-b-1-phosphomycoketide of M avium (E) A similar C34 derivative of M tuberculosis [121] The compounds in (D) and (E) are not related to polyisoprenoids, and are synthesized in pathogenic mycobacteria by a polyketide synthase [108] 2700 FEBS Journal 275 (2008) 2691–2711 ª 2008 The Author Journal compilation ª 2008 FEBS A role for the D-arabinose lipid carrier B A Wolucka Fig The last step of the biosynthesis of b-D-arabinofuranosyl-1-monophosphodecaprenol in mycobacteria (A) and D-erythroascorbic acid in yeasts (B) The oxidoreductase subunit (Rv3790) of the decaprenyl-phospho-ribose 2¢-epimerase of Mycobacterium tuberculosis shares 22% identity with the D-arabinono-1,4-lactone oxidase of Saccharomyces cerevisiae (ALO1); the enzymes catalyse similar reactions and employ structurally similar sugar intermediates of some eukaryotic microorganisms, such as trypanosomes, but also in plants Sequences similar to the mycobacterial enzymes of the decaprenyl-phosphoarabinose biosynthesis are apparently absent from eukaryotes, thus pointing to the existence of a totally different pathway(s) for the synthesis of d-arabinopyranosyl residues In agreement, GDP-d-arabinopyranose is the precursor of d-arabinopyranose residues present in the glycoconjugates of some trypanosomatid parasites In Leishmania major and Crithidia fasciculata, GDP-d-arabinose for the synthesis of lipophosphoglycan is synthesized from d-glucose via an undefined pathway that involves the loss of carbon C1 [63] Decaprenyl phosphate: structure and biosynthesis x,mono-E,octa-Z C50-decaprenyl phosphate (Fig 1A) [33] is the lipid carrier of mycobacteria that plays a crucial role in the biosynthesis of all three polymers of the cell wall: peptidoglycan, arabinogalactan and (lipo)arabinomannans As in many other eubacteria, plant chloroplasts, algae and apicomplexan parasites, mycobacteria synthesize isopentenyl diphosphate, the precursor for the biosynthesis of polyisoprenols and other isoprenoids, via the non-mevalonate (or 1-deoxy-d-xylulose-5-phosphate) route [64,65] Like the decaprenyl-phospho-arabinose pathway, non-mevalonate isoprenoid biosynthesis is a potential target for new antimycobacterial drugs [66,67] Polyisoprenyl phosphates are synthesized by sequential condensation of isopentenyl diphosphate with allylic diphosphates in a reaction catalysed by unrelated E- and Z-prenyl diphosphate synthases that introduce an E- and Z-isoprene unit, respectively, in the reaction product (Fig 3) (for a review, see [68]) In contrast with most bacteria that use C55-undecaprenol phosphate, consisting of 11 isoprene units in the x, di-E,octa-Z configuration, mycobacteria employ a shorter derivative: C50-decaprenyl phosphate [69] The mycobacterial decaprenol has a unique x,monoE,octa-Z stereoconfiguration of the polyisoprene chain (Fig 1A) [33] Such a structure implies the existence in mycobacteria of an unusual Z-prenyl diphosphate synthase that uses x,E-geranyl pyrophosphate (C10) and ⁄ or x,E,Z-farnesyl pyrophosphate (C15) as allylic substrate, instead of the common x,E,E-farnesyl pyrophosphate (Fig 3) The first Z-prenyl diphosphate synthase (Z-undecaprenyl diphosphate synthase) was identified in Micrococcus luteus [70] Mycobacterium tuberculosis contains two homologues of the M luteus Z-prenyl diphosphate synthase: the Rv1086 and Rv2361 proteins The Rv1086 gene is apparently not essential and encodes a specific, short-chain Z-farnesyl diphosphate synthase which synthesizes x,E,Z-farnesyl diphosphate and x,Z,Z-farnesyl diphosphate [71] (Fig 3) Another gene (Rv2361) encodes a Z-decaprenyl diphosphate synthase that preferentially uses x,E,Z-farnesyl diphosphate as a substrate [72] x,E-Geranyl diphosphate also serves as a substrate for the mycobacterial Z-decaprenyl FEBS Journal 275 (2008) 2691–2711 ª 2008 The Author Journal compilation ª 2008 FEBS 2701 A role for the D-arabinose lipid carrier B A Wolucka diphosphate synthase, albeit with lower efficiency This suggests that the Rv2361 Z-decaprenyl diphosphate synthase could compensate for the lack of Z-farnesyl diphosphate synthase activity in the Rv1086-deficient mutants of M tuberculosis (Fig 3) The mycobacterial Z-diphosphate synthases Rv1086 and Rv2361 not use dimethylallyl diphosphate as substrate Therefore, another, still unidentified, enzyme must exist that synthesizes either x,E-geranyl diphosphate or x,Z-neryl diphosphate in mycobacteria In addition, a novel, essential x,E,E-farnesyl diphosphate synthase (Rv3398) has been identified in M tuberculosis [73] The Rv3398 enzyme may be involved in the synthesis of compounds other than decaprenol isoprenoids, such as menaquinones, carotenoids or hopanoids, but its physiological function in mycobacteria is not known Interestingly, a non-essential bacA decaprenyl pyrophosphate phosphatase of M smegmatis (erroneously named ‘undecaprenyl phosphokinase’), a homologue of the Rv2136 protein of M tuberculosis, has been shown to be involved in biofilm formation and, not surprisingly, bacitracin resistance in M smegmatis [74] The bacA gene product participates in the recycling of polyprenyl pyrophosphates for cell-wall synthesis in bacteria The bacA deletion mutant of M smegmatis was viable, thus pointing to the existence of alternative pathways for the regeneration of decaprenyl phosphate in mycobacteria Chemically synthesized D-arabinose donors and analogues [14C]-Labelled b-d-arabinofuranosyl-1-monophosphodecaprenol was first obtained by a semi-in vivo micromethod [33], and served for the development of a basic assay for the mycobacterial arabinosyltransferases [75,76] The stereoselective chemical synthesis of b-d-[1-14C]arabinofuranosyl-1-monophosphodecaprenol was achieved using phosphoramidite coupling of decaprenol (of plant origin) and a protected 2,3,5-triO-tert-butyl dimethylsilyl derivative of Araf, followed by final deprotection with ammonium fluoride under mild conditions [77] A similar approach was used for the efficient synthesis of b-d-ribofuranosyl-1-monophosphodecaprenol and shorter chain C10-neryl- and C15-farnesyl-monophospho-b-d-ribofuranose derivatives [78] The b-dribofuranosyl-1-monophosphodecaprenol [34], a direct precursor of decaprenyl-phospho-arabinose, is necessary to study the decaprenyl-phospho-ribose 2¢-epimerase of mycobacteria Recently, it has been shown that the classical polyprenyl trichloroacetimidate-based methodology is 2702 particularly suitable for the stereoselective synthesis of polyprenyl-phospho-b-d-arabinofuranoses by coupling a polyprenyl trichloroacetimidate intermediate with a protected b-d-arabinofuranose 1-phosphate derivative [79] Studies of mycobacterial arabinosyltransferases require specific oligosaccharide acceptors, in addition to the b-d-arabinofuranosyl-1-monophosphodecaprenol donor A variety of O- and S-alkyl arabinosides were synthesized and tested as substrates in arabinosyltransferase assays [80–82] The chemically synthesized trisaccharide acceptors and the O-alkyl disaccharide acceptors with a C8 alkyl chain were good substrates for the mycobacterial arabinosyltransylferases, whereas monosaccharides did not serve as acceptors [80] Modified oligosaccharide analogues can inhibit polysaccharide synthesis, and may represent lead compounds for the synthesis of new drugs [81] Recently, fluorescent dansyl derivatives of Arafur(a1 fi 5)Arafur disaccharides were prepared as photoaffinity probes to study mycobacterial arabinosyltransferases and to screen drug candidates [83,84] Arabinosyltransferases, arabinan biosynthesis and the mode of action of ethambutol Recent studies have confirmed that decaprenyl-phospho-arabinose is the only donor of the arabinofuranosyl residues for the synthesis of the cell-wall arabinogalactans of Corynebacterineae [29,60] The synthetic b-d-arabinofuranosyl-1-monophosphodecaprenol supported the in vitro formation of a(1 fi 5) and b(1 fi 2) linkages of arabinans by a crude preparation of mycobacterial arabinosyltransferases [80] Further studies led to the identification of specific arabinosyltransferases, such as the priming AftA arabinosyltransferase (Rv3792), which adds the first arabinofuranosyl residue to the preformed linear galactan [60], and the terminal b(1 fi 2) AftB arabinosyltransferase (Rv3805), which is involved in the synthesis of mycolylation sites [85] Another arabinosyltransferase activity involved in the elaboration of arabinan chains of lipoarabinomannans has been detected recently, but not identified [86] Disruption of the Rv3806 orthologue gene encoding the decaprenyl-phospho-ribose-5¢-phosphate synthase [28], or of the priming AftA arabinosyltransferase [60], resulted in viable C glutamicum mutants that lacked both the arabinan part and the esterified corynomycolates of the cell-wall peptidoglycan–arabinogalactan– corynomycolate core These astonishing results demonstrate that, unlike mycobacteria, corynebacteria FEBS Journal 275 (2008) 2691–2711 ª 2008 The Author Journal compilation ª 2008 FEBS B A Wolucka not require arabinans for survival It might be envisaged, therefore, that arabinans play a distinct, although still unknown, role in mycobacteria, in addition to their structural functions Consequently, the regulation of arabinan synthesis in mycobacteria is expected to differ from that in corynebacteria In agreement, decaprenyl-phospho-arabinose does not accumulate in the disruption Cg-Emb mutant of C glutamicum [60], although it is accumulated in ethambutol-treated M smegmatis cells [32,33] Ethambutol, dextro-2,2¢-(ethylenediimino)-di-1-butanol, is a first-line antituberculosis drug with pleiotropic effects One of the early effects of ethambutol is the inhibition of arabinan biosynthesis in mycobacteria [87] Although ethambutol does not block the synthesis of decaprenyl-phospho-arabinose, it interferes with the utilization of the arabinose donor by inhibiting either arabinosyltransferase activity or the formation of an arabinose acceptor in mycobacteria [32,33] (Scheme 1, Fig 2) Studies of the mechanisms of resistance to ethambutol in M tuberculosis led to the identification of the embCAB operon [88] Structural mutations in embB and embC genes are found in clinical isolates of M tuberculosis [89], and the emb region is thought to determine intrinsic and acquired resistance to ethambutol in mycobacteria [90], or even broad drug resistance [91] The emb region of M tuberculosis encodes large (about 1000 amino acids) integral membrane proteins (EmbB, EmbA and EmbC) that share 61–68% sequence identity The Emb protein sequences are unique to Corynebacterineae and closely related species, and are involved in the formation of arabinan chains of the mycobacterial arabinogalactan (EmbB and EmbA) [92] and lipoarabinomannan (EmbC) [93] In contrast with M smegmatis, the embA gene is apparently essential in M tuberculosis, and expressed independently of the embC gene [94] Truncated forms of lipoarabinomannan were found in clinical isolates of ethambutol-resistant M tuberculosis [95] An early claim that the EmbAB proteins act as simple arabinosyltransferases [75] is now being revised Indeed, Emb proteins show little homology with known glycosyltransferases [96], and the arabinosyltransferase activity of Emb proteins has not been demonstrated in an unequivocal manner Recent studies have shown that ethambutol does not inhibit any of the identified arabinosyltransferases [60,85] Significantly, disruption of the emb gene results in l-glutamate efflux in C glutamicum [31] In the same line of evidence, ethambutol treatment results not only in a block of arabinan synthesis, but also in the loss of the previously formed arabinan from the cell wall in M smegmatis [97] A role for the D-arabinose lipid carrier However, the underlying mechanism is not understood The expression of embCAB genes in M tuberculosis undergoes a complex control process that involves the EmbR transcriptional regulator, and PknH [30] and other serine–threonine protein kinases ⁄ phosphatase systems [98] The serine–threonine protein kinase enzymes are absent from the M smegmatis saprophyte Therefore, significant differences in the regulation of Emb-dependent arabinan synthesis probably exist between the pathogenic and non-pathogenic mycobacteria In conclusion, a working hypothesis is that Emb proteins might act as arabinan-forming ‘polymerases’ or arabinan synthases that assemble larger blocks of oligosaccharide nature, but also function in substrate channelling and, perhaps, in species-specific signal transduction Clearly, the biological functions of Emb proteins and the actual target of ethambutol still await elucidation New drugs: decaprenyl-phosphoarabinose as a lead compound An amazing number of compounds with antimycobacterial activity have been designed, but only a few candidates, such as nitroimidazole PA-824 prodrug [99] and diarylquinolines [100], are currently undergoing clinical trials as antituberculosis drugs (for a review, see [101]) b-d-Arabinofuranosyl-1-monophosphodecaprenol [33] has served as a model molecule for the rational design of new antituberculosis drugs Of the tested phosphonate, phosphinic and sulfone analogues of decaprenylphospho-arabinose, a C-phosphonate analogue [102,103] is active against M tuberculosis and is currently undergoing trials in a mice model of tuberculosis Recently, new 2-deoxy-2-fluoro derivatives were obtained [104], as well as an aza-ribose analogue with promising antimycobacterial activity [105] Interestingly, an ethambutol-like diaminated compound SQ109, which contains two isoprene units, is a very efficient antimycobacterial, effective against multidrug-resistant strains (see [101]) Decaprenyl-phospho-D-mannose and related compounds Early studies on the mycobacterial decaprenyl-phospho-mannose played an important role in the discovery of d-arabinose-containing polyisoprenoid lipids and in a better understanding of arabinogalactan and FEBS Journal 275 (2008) 2691–2711 ª 2008 The Author Journal compilation ª 2008 FEBS 2703 A role for the D-arabinose lipid carrier B A Wolucka arabinomannan biosynthesis Two forms of polyprenyl-phospho-mannose were synthesized from GDP[14C]mannose by membrane fractions of M smegmatis: a C50-decaprenyl-phospho-mannose and a C35-octahydroheptaprenyl-phospho-mannose [69,106] Similar endogenously synthesized activated mannose derivatives were isolated from M smegmatis and structurally characterized as b-d-mannopyranosyl-1-monophosphodecaprenol (Fig 1D) and b-d-mannopyranosyl-1monophospho-C35-octahydroheptaprenol (Fig 4A) [107] Surprisingly, the short-chain C35-octahydroheptaprenyl-phospho-mannose was the predominant form, whereas decaprenyl-phospho-mannose represented only 5% of the total polyprenyl-phospho-monosaccharides of M smegmatis Although decaprenyl phosphate is a lipid carrier in M tuberculosis and other mycobacteria, the partially saturated C35-octahydroheptaprenol was found only in the saprophytic M smegmatis species, and its synthesis is totally unknown The saturated isoprenoid-like x-end of the lipid chain is similar to the recently identified mycoketides of M tuberculosis [108] However, the presence of unsaturated isoprene residues next to the a-hydroxyl group points to an isoprenoid route of lipid synthesis Perhaps, the C35-octahydroheptaprenol is synthesized via a convergent isoprene and polyketide biosynthetic machinery similar to that described in Bacillus subtilis [109] Otherwise, the C35-octahydroheptaprenol might be synthesized from an unsaturated heptaprenyl diphosphate intermediate [110] The enzymes involved in the synthesis and biological functions of C35-octahydroheptaprenyl-phospho-mannose in M smegmatis are still unknown b-d-Mannosyl-1-phosphodecaprenol is synthesized from decaprenyl phosphate and GDP-mannose by a GDP-mannose-dependent mannosyltransferase Ppm1 (Rv2051) [111,112] Decaprenyl-phospho-mannose is a substrate for mannosyltransferases that are involved in the synthesis of phosphatidylinositolmannosides, lipomannans, lipoarabinomannans and glycoproteins in mycobacteria A decaprenyl-phospho-mannose-dependent mannosyltransferase PimE (Rv1159) has been shown to synthesize the PIM5 phosphatidylinositolmannoside [113] Another enzyme, a branching a1, 2-mannosyltransferase (Rv2181), is necessary for the synthesis of lipomannan [114] Inactivation of the Rv2181 orthologue resulted in the lack of lipomannan and the formation of a truncated lipoarabinomannan in M smegmatis, thus suggesting that the two polymers are synthesized via at least partially independent routes In addition, mannosyltransferases involved in the extension of the lipomannan ⁄ lipoarabinomannan core precursor (Rv2174) [115], the synthesis of man2704 nose caps of lipoarabinomannan (Rv1635) [116] and protein O-mannosylation (Rv1002) [117] have been identified as putative decaprenyl-phospho-mannosedependent enzymes Decaprenyl phosphate is also a carrier of building blocks for peptidoglycan synthesis [118], and participates in the synthesis of lipid-linked pyrophosphoryloligosaccharides in the assembly of the peptidoglycan–galactan part of the mycobacterial cell wall [119] The ‘apparent carrier’ for mycolic acids of M smegmatis From the lipid extracts of M smegmatis, Besra et al [120] isolated a mycolic acid ester of C35-octahydroheptaprenyl-phospho-mannose (Myc-PL): 6-O-mycolyl-b-d-mannopyranosyl-1-monophosphoryl-3,7,11,15, 19,23,27-heptamethyl-(2Z,6E,10E)-octacosatrien-1-ol (Fig 4C) It is worth noting that the stereoconfiguration of the isoprene units (E,E,Z-a) in the structure proposed in [120] differs from that determined for the b-d-mannosyl-1-monophospho-octahydroheptaprenol of M smegmatis (Z,Z,Z-a) (Fig 4A) [107] Moreover, the b-d-mannosyl-1-monophospho-octahydroheptaprenol was shown to be the major form of lipid-linked mannose, and present in a non-esterified state in M smegmatis [107] It is possible that the latter discrepancy is a result of the use of different procedures for lipid isolation In particular, spontaneous or enzyme-mediated acyl migration might occur as a result of the alkaline conditions applied in [120] Another possibility is that the esterification step does not occur immediately after b-d-mannosyl-1-monophospho-octahydroheptaprenol synthesis, but only later during ageing of M smegmatis cells C35-Octahydroheptaprenol phosphate derivatives have never been found in M tuberculosis and other pathogenic mycobacteria [106,121] Therefore, the mycoloylated mannophospholipid of M smegmatis (Fig 4C), described in [120], is obviously not a common carrier for mycolic acids in mycobacteria Mycobacterial ‘polyisoprenoid glycolipids’, phosphoantigens and immune response Human T cells of the immune system recognize peptide antigens, but also respond to lipid and glycolipid antigens displayed by CD1 proteins, and to some ill-defined phosphoantigens This recognition plays an important role in both innate and acquired immunity during tuberculosis infection FEBS Journal 275 (2008) 2691–2711 ª 2008 The Author Journal compilation ª 2008 FEBS B A Wolucka Human CD1c proteins recognize fully saturated (C30–C34) mannosyl-b-1-phospholipids, erroneously called ‘polyisoprenoid glycolipids’, of pathogenic mycobacteria (Fig 4D,E), in addition to short-chain (C35) a-saturated mannosyl-b-1-phosphoryldolichols [121] The branched alkyl chain of the antigenic mannosyl-b-1-phospholipid of M tuberculosis is synthesized from malonyl (C2) and methylmalonyl (C3) units by the PKS12 polyketide synthase [108] Thus, in spite of some structural resemblance to polyisoprenols, the antigenic phosphoglycolipids of mycobacterial pathogens belong to a new class of secondary metabolites: phosphorylated and mannosylated polyketides (mannosyl-b-1-phosphomycoketides) Interestingly, the partially saturated (C35) b-d-mannosyl-1-phospho-octahydroheptaprenol of M smegmatis [107], but not a similar mycoloylated derivative Myc-PL [120], was recognized by human CD8 cells [121] Independent of CD1c-mediated recognition, mycobacterial non-peptide phosphoantigens, including isoprenoid products of the non-mevalonate pathway, are recognized by Vc2Vd2 T cells via a mechanism that does not require antigen processing or presentation by major histocompatibility complex I and II or CD1 molecules [122] However, the structures and mechanism of action of mycobacterial phosphoantigens are still a matter of debate [123] New methods for the analysis of polyisoprenoid glycolipids The application of tandem mass spectrometric methods for the analysis of polyisoprenoid glycolipids was first demonstrated using preparations of polyprenyl-phospho-sugars isolated from M smegmatis [61] Desorption chemical ionization tandem mass spectrometry proved to be suitable for the structural determination of polyisoprenyl phosphates and, in particular, allowed a facile discrimination between unsaturated polyprenols and a-saturated (dolichol) derivatives [124] Recent developments in related desorption electrospray ionization methods for ambient analysis of complex solid-state samples [125] will probably find new applications in the field of lipidomics and metabolomics of isoprenoid compounds Fast-atom bombardment and, later, electrosprayionization tandem mass spectrometric techniques have been shown to be useful for the determination of the anomeric configuration of the glycosyl residue of polyisoprenyl-phospho-sugars, sugar nucleotides and sugar 1-phosphates [126,127] Collision-induced dissociation of glycosyl 1-phosphate derivatives produced different fragmentation patterns depending on the cis ⁄ trans A role for the D-arabinose lipid carrier configuration of their 2-hydroxyl and phosphate groups Further studies have shown that stereochemistry at the 2-position of the non-reducing sugar ring affects the fragmentation of disaccharides [128] and acetyl glycosides [129], thus allowing anomeric distinction of these non-phosphorylated derivatives The method was successfully applied for the determination of the anomeric linkage of the d-mannosyl residue of the scarce, antigenic C30–C34 mannosyl-b-1-phosphomycoketides (erroneously called ‘polyisoprenoid glycolipids’) isolated from M tuberculosis and M avium (Fig 4D,E) [121,130] A similar method was applied to screen for galactose 1-phosphate levels in neonatal galactosaemia [131], and for the identification of a novel sugar nucleotide precursor of pseudaminic acid of the Campylobacter jejuni pathogen [132] Conclusion The discovery of decaprenyl-phospho-d-arabinose and decaprenyl-phospho-ribose in 1990, and the subsequent proposal of the last steps of the Araf pathway in 1992 (Scheme 1) [32], marked the beginning of a new era in the study of cell-wall synthesis in mycobacteria The complete pathway to d-arabinose proposed here is unique in bacteria and represents a good target for new drugs The enzymes for d-arabinose biosynthesis have not been studied and await thorough biochemical characterization The novel decaprenyl-phospho-ribose 2¢-epimerase and, in particular, its relatedness to l-ascorbic acid (vitamin C) biosynthetic enzymes, including the recently evoked GDP-d-mannose 2¢-epimerase [57], deserve special attention In addition, similar 2¢-epimerases may be involved in the biosynthesis of still unknown, water-soluble d-arabinofuranosyl donors for glycoconjugates, whose synthesis does not require long-chain polyisoprenyl carriers Ethambutol, a first-line antituberculosis drug, does not interfere with the synthesis of d-arabinose, but rather inhibits the incorporation of d-arabinofuranosyl residues of decaprenyl-phospho-arabinose into the arabinogalactan and (lipo)arabinomannan of the mycobacterial cell wall at the level of the Emb proteins – the putative arabinan synthases Specific mutations in the Emb proteins confer resistance to ethambutol in M tuberculosis [88] However, in spite of almost 15 years of intensive research all over the world, and our better knowledge of the structure, functions and biosynthesis of the mycobacterial cell wall, the mode of action of ethambutol and the molecular mechanisms of resistance to the drug are not understood FEBS Journal 275 (2008) 2691–2711 ª 2008 The Author Journal compilation ª 2008 FEBS 2705 A role for the D-arabinose lipid carrier B A Wolucka However, synthetic isoprenoid derivatives, including those based on the structure of b-d-arabinofuranosylmonophospho-decaprenol and b-d-ribofuranosyl-monophospho-decaprenol, have promising antituberculosis activity, and some may show immunomodulatory effects Further research by new generations of enthusiastic and devoted scientists will be needed to take up the challenge 13 14 References Dye C (2006) Global epidemiology of tuberculosis Lancet 367, 938–940 Nunn P, Williams B, Floyd K, Dye C, Elzinga G & Raviglione M (2005) Tuberculosis control in the era of HIV Nat Rev Immunol 5, 819–826 Gandhi NR, Moll A, Sturm AW, Pawinski R, Govender T, Lalloo U, Zeller K, Andrews J & Friedland G (2006) Extensively drug-resistant tuberculosis as a cause of death in patients co-infected with tuberculosis and HIV in a rural area of South Africa Lancet 368, 1575–1580 Raviglione MC & Smith IM (2007) XDR tuberculosis – implications for global public health N Engl J Med 356, 656–659 Coley J, Tarelli E, Archibald AR & Baddiley J (1978) The linkage between teichoic acid and peptidoglycan in bacterial cell walls FEBS Lett 88, 1–9 Brennan PJ (2003) Structure, function, and biogenesis of the cell wall of Mycobacterium tuberculosis Tuberculosis (Edinb) 83, 91–97 van der Wel N, Hava D, Houben D, Fluitsma D, van Zon M, Pierson J, Brenner M & Peters PJ (2007) M tuberculosis and M leprae translocate from the phagolysosome to the cytosol in myeloid cells Cell 129, 1287–1298 Ortalo-Magne A, Dupont MA, Lemassu A, Andersen AB, Gounon P & Daffe M (1995) Molecular composition of the outermost capsular material of the tubercle bacillus Microbiology 141, 1609–1620 Trias J, Jarlier V & Benz R (1992) Porins in the cell wall of mycobacteria Science 258, 1479–1481 10 Niederweis M (2003) Mycobacterial porins – new channel proteins in unique outer membranes Mol Microbiol 49, 1167–1177 11 Zhang Y, Heym B, Allen B, Young D & Cole S (1992) The catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis Nature 358, 591–593 12 Rosenkrands I, Weldingh K, Jacobsen S, Hansen CV, Florio W, Gianetri I & Andersen P (2000) Mapping and identification of Mycobacterium tuberculosis proteins by two-dimensional gel electrophoresis, microse- 2706 15 16 17 18 19 20 21 22 23 quencing and immunodetection Electrophoresis 21, 935–948 De Bruyn J, Soetaert K, Buyssens P, Calonne I, De Coene JL, Gallet X, Brasseur R, Wattiez R, Falmagne P, Montrozier H et al (2000) Evidence for specific and non-covalent binding of lipids to natural and recombinant Mycobacterium bovis BCG hsp60 proteins, and to the Escherichia coli homologue GroEL Microbiology 146, 1513–1524 Ojha A, Anand M, Bhatt A, Kremer L, Jacobs WR & Hatfull GF (2005) GroEL1: a dedicated chaperone involved in mycolic acid biosynthesis during biofilm formation in mycobacteria Cell 123, 861–873 Belisle JT, Vissa VD, Sievert T, Takayama K, Brennan PJ & Besra GS (1997) Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis Science 276, 1420–1422 Katsube T, Matsumoto S, Takatsuka M, Okuyama M, Ozeki Y, Naito M, Nishiuchi Y, Fujiwara N, Yoshimura M, Tsuboi T et al (2007) Control of cell wall assembly by a histone-like protein in mycobacteria J Bacteriol 189, 8241–8249 Harth G, Zamecnik PC, Tang JY, Tabatadze D & Horwitz MA (2000) Treatment of Mycobacterium tuberculosis with antisense oligonucleotides to glutamine synthetase mRNA inhibits glutamine synthetase activity, formation of the poly-l-glutamate ⁄ glutamine cell wall structure, and bacterial replication Proc Natl Acad Sci USA 97, 418–423 Walburger A, Koul A, Ferrari G, Nguyen L, Prescianotto-Baschong C, Huygen K, Klebl B, Thompson C, Bacher G & Pieters J (2004) Protein kinase G from pathogenic mycobacteria promotes survival within macrophages Science 304, 1800–1804 Jones G & Dyson P (2006) Evolution of transmembrane protein kinases implicated in coordinating remodeling of gram-positive peptidoglycan: inside versus outside J Bacteriol 188, 7470–7476 Narayan A, Sachdeva P, Sharma K, Saini AK, Tyagi AK & Singh Y (2007) Serine threonine protein kinases of mycobacterial genus: phylogeny to function Physiol Genomics 29, 66–75 Brodin P, Rosenkrands I, Andersen P, Cole ST & Brosch R (2004) ESAT-6 proteins: protective antigens and virulence factors? Trends Microbiol 12, 500–508 Basu SK, Kumar D, Singh DK, Ganguly N, Siddiqui Z, Rao KV & Sharma P (2006) Mycobacterium tuberculosis secreted antigen (MTSA-10) modulates macrophage function by redox regulation of phosphatases Febs J 273, 5517–5534 Seiler P, Ulrichs T, Bandermann S, Pradl L, Jorg S, Krenn V, Morawietz L, Kaufmann SH & Aichele P (2003) Cell-wall alterations as an attribute of FEBS Journal 275 (2008) 2691–2711 ª 2008 The Author Journal compilation ª 2008 FEBS B A Wolucka 24 25 26 27 28 29 30 31 32 33 Mycobacterium tuberculosis in latent infection J Infect Dis 188, 1326–1331 Neyrolles O, Hernandez-Pando R, Pietri-Rouxel F, Fornes P, Tailleux L, Barrios Payan JA, Pivert E, Bordat Y, Aguilar D, Prevost MC et al (2006) Is adipose tissue a place for Mycobacterium tuberculosis persistence? PLoS ONE 1, e43 Alteri CJ, Xicohtencatl-Cortes J, Hess S, CaballeroOlin G, Giron JA & Friedman RL (2007) Mycobacterium tuberculosis produces pili during human infection Proc Natl Acad Sci USA 104, 5145–5150 Watanabe M, Ohta A, Sasaki SI & Minnikin D (1999) Structure of a new glycolipid from the Mycobacterium avium–Mycobacterium intracellulare complex J Bacteriol 181, 2293–2297 Chatterjee D & Khoo KH (1998) Mycobacterial lipoarabinomannan: an extraordinary lipoheteroglycan with profound physiological effects Glycobiology 8, 113–120 Alderwick LJ, Radmacher E, Seidel M, Gande R, Hitchen PG, Morris HR, Dell A, Sahm H, Eggeling L & Besra GS (2005) Deletion of Cg-emb in Corynebacterineae leads to a novel truncated cell wall arabinogalactan, whereas inactivation of Cg-ubiA results in an arabinan-deficient mutant with a cell wall galactan core J Biol Chem 280, 32362–32371 Alderwick LJ, Dover LG, Seidel M, Gande R, Sahm H, Eggeling L & Besra GS (2006) Arabinan-deficient mutants of Corynebacterium glutamicum and the consequent flux in decaprenylmonophosphoryld-arabinose metabolism Glycobiology 16, 1073– 1081 Molle V, Kremer L, Girard-Blanc C, Besra GS, Cozzone AJ & Prost JF (2003) An FHA phosphoprotein recognition domain mediates protein EmbR phosphorylation by PknH, a Ser ⁄ Thr protein kinase from Mycobacterium tuberculosis Biochemistry 42, 15300– 15309 Radmacher E, Stansen KC, Besra GS, Alderwick LJ, Maughan WN, Hollweg G, Sahm H, Wendisch VF & Eggeling L (2005) Ethambutol, a cell wall inhibitor of Mycobacterium tuberculosis, elicits l-glutamate efflux of Corynebacterium glutamicum Microbiology 151, 1359–1368 Wolucka BA (1992) D-Arabinose metabolism in mycobacteria Isolation of two novel glycolipids from Mycobacterium smegmatis PhD Thesis University of Louvain, Louvain-la-Neuve Wolucka BA, McNeil MR, de HoffmannE, Chojnacki T & Brennan PJ (1994) Recognition of the lipid intermediate for arabinogalactan ⁄ arabinomannan biosynthesis and its relation to the mode of action of ethambutol on mycobacteria J Biol Chem 269, 23328– 23335 A role for the D-arabinose lipid carrier 34 Wolucka BA & de Hoffmann E (1995) The presence of beta-d-ribosyl-1-monophosphodecaprenol in mycobacteria J Biol Chem 270, 20151–20155 35 McNeil MR & Brennan PJ (1991) Structure, function and biogenesis of the cell envelope of mycobacteria in relation to bacterial physiology, pathogenesis and drug resistance; some thoughts and possibilities arising from recent structural information Res Microbiol 142, 451– 463 36 Scherman M, Weston A, Duncan K, Whittington A, Upton R, Deng L, Comber R, Friedrich JD & McNeil M (1995) Biosynthetic origin of mycobacterial cell wall arabinosyl residues J Bacteriol 177, 7125–7130 37 Scherman MS, Kalbe-Bournonville L, Bush D, Xin Y, Deng L & McNeil M (1996) Polyprenylphosphate-pentoses in mycobacteria are synthesized from 5-phosphoribose pyrophosphate J Biol Chem 271, 29652– 29658 38 Izumori K, Watanabe Y & Sugimoto S (1980) Evolution of d-arabinose, l-xylose and l-ribose utilization in Mycobacterium smegmatis – mutants with a novel enzyme pentose reductase Agric Biol Chem 44, 1443– 1446 39 Wolucka-Wojtkiewicz B, Szmidzinski R, Jezierska A & Cocito C (1988) Identification of a salvage pathway for d-arabinose in Mycobacterium smegmatis Eur J Biochem 172, 197–203 40 Chiang C & Knight SG (1960) Metabolism of d-xylose by moulds Nature 188, 79–81 41 Watanabe S, Kodaki T & Makino K (2006) Cloning, expression, and characterization of bacterial l-arabinose 1-dehydrogenase involved in an alternative pathway of l-arabinose metabolism J Biol Chem 281, 2612–2623 42 Brouns SJ, Walther J, Snijders AP, van de Werken HJ, Willemen HL, Worm P, de Vos MG, Andersson A, Lundgren M, Mazon HF et al (2006) Identification of the missing links in prokaryotic pentose oxidation pathways: evidence for enzyme recruitment J Biol Chem 281, 27378–27388 43 Amako K, Fujita K, Iwamoto C, Sengee M, Fuchigami K, Fukumoto J, Ogishi Y, Kishimoto R & Goda K (2006) NADP(+)-dependent d-arabinose dehydrogenase shows a limited contribution to erythroascorbic acid biosynthesis and oxidative stress resistance in Saccharomyces cerevisiae Biosci Biotechnol Biochem 70, 3004–3012 44 Kim ST, Huh WK, Lee BH & Kang SO (1998) d-arabinose dehydrogenase and its gene from Saccharomyces cerevisiae Biochim Biophys Acta 1429, 29–39 45 Lechner J, Wieland F & Sumper M (1985) Biosynthesis of sulfated saccharides N-glycosidically linked to the protein via glucose Purification and identification of FEBS Journal 275 (2008) 2691–2711 ª 2008 The Author Journal compilation ª 2008 FEBS 2707 A role for the D-arabinose lipid carrier 46 47 48 49 50 51 52 53 54 55 56 B A Wolucka sulfated dolichyl monophosphoryl tetrasaccharides from halobacteria J Biol Chem 260, 860–866 Trent MS, Ribeiro AA, Doerrler WT, Lin S, Cotter RJ & Raetz CR (2001) Accumulation of a polyisoprene-linked amino sugar in polymyxin-resistant Salmonella typhimurium and Escherichia coli: structural characterization and transfer to lipid A in the periplasm J Biol Chem 276, 43132–43144 Kanjilal-Kolar S & Raetz CR (2006) Dodecaprenyl phosphate-galacturonic acid as a donor substrate for lipopolysaccharide core glycosylation in Rhizobium leguminosarum J Biol Chem 281, 12879–12887 Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon SV, Eiglmeier K, Gas S, Barry CE 3rd et al (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence Nature 393, 537–544 Cole ST, Eiglmeier K, Parkhill J, James KD, Thomson ´ NR, Wheeler PR, Honore N, Garnier T, Churcher C, Harris D et al (2001) Massive gene decay in the leprosy bacillus Nature 409, 1007–1011 Mikusova K, Huang H, Yagi T, Holsters M, Vereecke D, D’Haeze W, Scherman MS, Brennan PJ, McNeil MR & Crick DC (2005) Decaprenylphosphoryl arabinofuranose, the donor of the d-arabinofuranosyl residues of mycobacterial arabinan, is formed via a two-step epimerization of decaprenylphosphoryl ribose J Bacteriol 187, 8020–8025 Huang H, Scherman MS, D’Haeze W, Vereecke D, Holsters M, Crick DC & McNeil MR (2005) Identification and active expression of the Mycobacterium tuberculosis gene encoding 5-phospho-{alpha}-d-ribose1-diphosphate:decaprenyl-phosphate 5-phosphoribosyltransferase, the first enzyme committed to decaprenylphosphoryl-d-arabinose synthesis J Biol Chem 280, 24539–24543 Sassetti CM, Boyd DH & Rubin EJ (2003) Genes required for mycobacterial growth defined by high density mutagenesis Mol Microbiol 48, 77–84 Eriksen TA, Kadziola A, Bentsen AK, Harlow KW & Larsen S (2000) Structural basis for the function of Bacillus subtilis phosphoribosyl-pyrophosphate synthetase Nat Struct Biol 7, 303–308 Forbes M, Kuck NA & Peets EA (1965) Effect of ethambutol on nucleic acid metabolism in Mycobacterium smegmatis and its reversal by polyamines and divalent cations J Bacteriol 89, 1299–1305 Mergaert P, Van Montagu M, Prome JC & Holsters M (1993) Three unusual modifications, a d-arabinosyl, an N-methyl, and a carbamoyl group, are present on the Nod factors of Azorhizobium caulinodans strain ORS571 Proc Natl Acad Sci USA 90, 1551– 1555 Wolucka BA & van Montagu M (2003) GDP-mannose 3¢,5¢-epimerase forms GDP-l-gulose, a putative 2708 57 58 59 60 61 62 63 64 65 66 67 68 intermediate for the de novo biosynthesis of vitamin C in plants J Biol Chem 278, 47483–47490 Wolucka BA & van Montagu M (2007) The VTC2 cycle and the de novo biosynthesis pathways for vitamin C in plants Phytochemistry 68, 2602–2613 Huh WK, Lee BH, Kim ST, Kim YR, Rhie GE, Baek YW, Hwang CS, Lee JS & Kang O (1998) d-Erythroascorbic acid is an important antioxidant molecule in Saccharomyces cerevisiae Mol Microbiol 30, 895–903 Wolucka BA & Communi D (2006) Mycobacterium tuberculosis possesses a functional enzyme for the synthesis of vitamin C, l-gulono-1,4-lactone dehydrogenase Febs J 273, 4435–4445 Alderwick LJ, Seidel M, Sahm H, Besra GS & Eggeling L (2006) Identification of a novel arabinofuranosyltransferase (AftA) involved in cell wall arabinan biosynthesis in Mycobacterium tuberculosis J Biol Chem 281, 15653–15661 Wolucka BA & de Hoffmann E (1994) Mass spectrometric analysis of prenyl phosphates and their glycosylated forms Acta Biochim Pol 41, 345–349 Tatituri RV, Alderwick LJ, Mishra AK, Nigou J, Gilleron M, Krumbach K, Hitchen P, Giordano A, Morris HR, Dell A et al (2007) Structural characterization of a partially arabinosylated lipoarabinomannan variant isolated from a Corynebacterium glutamicum ubiA mutant Microbiology 153, 2621– 2629 Schneider P, McConville MJ & Ferguson MA (1994) Characterization of GDP-alpha-d-arabinopyranose, the precursor of d-Arap in Leishmania major lipophosphoglycan J Biol Chem 269, 18332–18337 Rohmer M, Knani M, Simonin P, Sutter B & Sahm H (1993) Isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate Biochem J 295, 517–524 Rohmer M (1999) The discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria, algae and higher plants Nat Prod Rep 16, 565–574 Kuntz L, Tritsch D, Grosdemange-Billiard C, Hemmerlin A, Willem A, Bach TJ & Rohmer M (2005) Isoprenoid biosynthesis as a target for antibacterial and antiparasitic drugs: phosphonohydroxamic acids as inhibitors of deoxyxylulose phosphate reducto-isomerase Biochem J 386, 127–135 Henriksson LM, Unge T, Carlsson J, Aqvist J, Mowbray SL & Jones TA (2007) Structures of Mycobacterium tuberculosis 1-deoxy-d-xylulose-5-phosphate reductoisomerase provide new insights into catalysis J Biol Chem 282, 19905–19916 Swiezewska E & Danikiewicz W (2005) Polyisoprenoids: structure, biosynthesis and function Progress Lipid Res 44, 235–258 FEBS Journal 275 (2008) 2691–2711 ª 2008 The Author Journal compilation ª 2008 FEBS B A Wolucka 69 Takayama K & Goldman DS (1970) Enzymatic synthesis of mannosyl-1-phosphoryl-decaprenol by a cellfree system of Mycobacterium tuberculosis J Biol Chem 245, 6251–6257 70 Shimizu N, Koyama T & Ogura K (1998) Molecular cloning, expression, and purification of undecaprenyl diphosphate synthase No sequence similarity between E- and Z-prenyl diphosphate synthases J Biol Chem 273, 19476–19481 71 Schulbach MC, Mahapatra S, Macchia M, Barontini S, Papi C, Minutolo F, Bertini S, Brennan PJ & Crick DC (2001) Purification, enzymatic characterization, and inhibition of the Z-farnesyl diphosphate synthase from Mycobacterium tuberculosis J Biol Chem 276, 11624–11630 72 Kaur D, Brennan PJ & Crick DC (2004) Decaprenyl diphosphate synthesis in Mycobacterium tuberculosis J Bacteriol 186, 7564–7570 73 Dhiman RK, Schulbach MC, Mahapatra S, Baulard AR, Vissa V, Brennan PJ & Crick DC (2004) Identification of a novel class of omega,E,E-farnesyl diphosphate synthase from Mycobacterium tuberculosis J Lipid Res 45, 1140–1147 74 Rose L, Kaufmann SH & Daugelat S (2004) Involvement of Mycobacterium smegmatis undecaprenyl phosphokinase in biofilm and smegma formation Microbes Infect 6, 965–971 75 Belanger AE, Besra GS, Ford ME, Mikusova K, Belisle JT, Brennan PJ & Inamine JM (1996) The embAB genes of Mycobacterium avium encode an arabinosyl transferase involved in cell wall arabinan biosynthesis that is the target for the antimycobacterial drug ethambutol Proc Natl Acad Sci USA 93, 11919–11924 76 Xin Y, Lee RE, Scherman MS, Khoo KH, Besra GS, Brennan PJ & McNeil M (1997) Characterization of the in vitro synthesized arabinan of mycobacterial cell walls Biochim Biophys Acta 1335, 231–234 77 Lee RE, Brennan PJ & Besra GS (1998) Synthesis of beta-d-arabinofuranosyl-1-monophosphoryl polyprenols: examination of their function as mycobacterial arabinosyl transferase donors Bioorg Med Chem Lett 8, 951–954 78 Liav A, Swiezewska E, Ciepichal E & Brennan PJ (2006a) Stereoselectivity in the synthesis of polyprenylphosphoryl b-d-ribofuranoses Tetrahedron Lett 47, 8781–8783 79 Liav A, Huang H, Ciepichal E, Brennan PJ & McNeil MR (2006b) Stereoselective synthesis of decaprenylphosphoryl b-d-arabinofuranose Tetrahedron Lett 47, 545–547 80 Lee RE, Brennan PJ & Besra GS (1997) Mycobacterial arabinan biosynthesis: the use of synthetic arabinoside acceptors in the development of an arabinosyl transfer assay Glycobiology 7, 1121–1128 A role for the D-arabinose lipid carrier 81 Ayers JD, Lowary TL, Morehouse CB & Besra GS (1998) Synthetic arabinofuranosyl oligosaccharides as mycobacterial arabinosyltransferase substrates Bioorg Med Chem Lett 8, 437–442 82 Pathak AK, Pathak V, Maddry JA, Suling WJ, Gurcha SS, Besra GS & Reynolds RC (2001) Studies on alpha(1 fi 5) linked octyl arabinofuranosyl disaccharides for mycobacterial arabinosyl transferase activity Bioorg Med Chem 9, 3145–3151 83 Pathak AK, Pathak V, Gurcha SS, Besra GS & Reynolds RC (2002) Synthesis of an arabinofuranosyl disaccharide photoaffinity probe for arabinosyltransferase activity in Mycobacterium tuberculosis Bioorg Med Chem Lett 12, 2749–2752 84 Pathak AK, Pathak V, Seitz L, Gurcha SS, Besra GS, Riordan JM & Reynolds RC (2007) Disaccharide analogs as probes for glycosyltransferases in Mycobacterium tuberculosis Bioorg Med Chem 15, 5629–5650 85 Seidel M, Alderwick LJ, Birch HL, Sahm H, Eggeling L & Besra GS (2007) Identification of a novel arabinofuranosyltransferase AftB involved in a terminal step of cell wall arabinan biosynthesis in Corynebacterineae, such as Corynebacterium glutamicum and Mycobacterium tuberculosis J Biol Chem 282, 14729– 14740 86 Zhang J, Khoo KH, Wu SW & Chatterjee D (2007) Characterization of a distinct arabinofuranosyltransferase in Mycobacterium smegmatis J Am Chem Soc 129, 9650–9662 87 Takayama K & Kilburn JO (1989) Inhibition of synthesis of arabinogalactan by ethambutol in Mycobacterium smegmatis Antimicrob Agents Chemother 33, 1493–1499 88 Telenti A, Philipp WJ, Sreevatsan S, Bernasconi C, Stockbauer KE, Wieles B, Musser JM & Jacobs WR (1997) The emb operon, a gene cluster of Mycobacterium tuberculosis involved in resistance to ethambutol Nat Med 3, 567–570 89 Srivastava S, Garg A, Ayyagari A, Nyati KK, Dhole TN & Dwivedi SK (2006) Nucleotide polymorphism associated with ethambutol resistance in clinical isolates of Mycobacterium tuberculosis Curr Microbiol 53, 401–405 90 Alcaide F, Pfyffer GE & Telenti A (1997) Role of embB in natural and acquired resistance to ethambutol in mycobacteria Antimicrob Agents Chemother 41, 2270–2273 91 Hazbon MH, Bobadilla del Valle M, Guerrero MI, Varma-Basil M, Filliol I, Cavatore M, Colangeli R, Safi H, Billman-Jacobe H, Lavender C et al (2005) Role of embB codon 306 mutations in Mycobacterium tuberculosis revisited: a novel association with broad drug resistance and IS6110 clustering rather than ethambutol resistance Antimicrob Agents Chemother 49, 3794–3802 FEBS Journal 275 (2008) 2691–2711 ª 2008 The Author Journal compilation ª 2008 FEBS 2709 A role for the D-arabinose lipid carrier B A Wolucka 92 Escuyer VE, Lety MA, Torrelles JB, Khoo KH, Tang JB, Rithner CD, Frehel C, McNeil MR, Brennan PJ & Chatterjee D (2001) The role of the embA and embB gene products in the biosynthesis of the terminal hexaarabinofuranosyl motif of Mycobacterium smegmatis arabinogalactan J Biol Chem 276, 48854– 48862 93 Zhang N, Torrelles JB, McNeil MR, Escuyer VE, Khoo KH, Brennan PJ & Chatterjee D (2003) The Emb proteins of mycobacteria direct arabinosylation of lipoarabinomannan and arabinogalactan via an N-terminal recognition region and a C-terminal synthetic region Mol Microbiol 50, 69–76 94 Amin AG, Goude R, Shi L, Zhang J, Chatterjee D & Parish T (2008) EmbA is an essential arabinosyltransferase in Mycobacterium tuberculosis Microbiology 154, 240–248 95 Torrelles JB, Khoo KH, Sieling PA, Modlin RL, Zhang N, Marques AM, Treumann A, Rithner CD, Brennan PJ & Chatterjee D (2004) Truncated structural variants of lipoarabinomannan in Mycobacterium leprae and an ethambutol-resistant strain of Mycobacterium tuberculosis J Biol Chem 279, 41227–41239 96 Berg S, Kaur D, Jackson M & Brennan PJ (2007) The glycosyltransferases of Mycobacterium tuberculosis – roles in the synthesis of arabinogalactan, lipoarabinomannan, and other glycoconjugates Glycobiology 17, 35–56 97 Dong X, Bhamidi S, Scherman M, Xin Y & McNeil MR (2006) Development of a quantitative assay for mycobacterial endogenous arabinase and ensuing studies of arabinase levels and arabinan metabolism in Mycobacterium smegmatis Appl Environ Microbiol 72, 2601–2605 98 Sharma K, Gupta M, Krupa A, Srinivasan N & Singh Y (2006) EmbR, a regulatory protein with ATPase activity, is a substrate of multiple serine ⁄ threonine kinases and phosphatase in Mycobacterium tuberculosis Febs J 273, 2711–2721 99 Stover CK, Warrener P, VanDevanter DR, Sherman DR, Arain TM, Langhorne MH, Anderson SW, Towell JA, Yuan Y, McMurray DN et al (2000) A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis Nature 405, 962–966 100 Andries K, Verhasselt P, Guillemont J, Gohlmann HW, Neefs JM, Winkler H, van Gestel J, Timmerman P, Zhu M, Lee E et al (2005) A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis Science 307, 223–227 101 Janin YL (2007) Antituberculosis drugs: ten years of research Bioorg Med Chem 15, 2479–2513 102 Centrone CA & Lowary TL (2002) Synthesis and antituberculosis activity of C-phosphonate analogues of decaprenolphosphoarabinose, a key intermediate in the biosynthesis of mycobacterial arabinogalactan 2710 103 104 105 106 107 108 109 110 111 112 113 and lipoarabinomannan J Org Chem 67, 8862– 8870 Centrone CA & Lowary TL (2004) Sulfone and phosphinic acid analogs of decaprenolphosphoarabinose as potential anti-tuberculosis agents Bioorg Med Chem 12, 5495–5503 Joe M & Lowary TL (2006) Synthesis of 2-deoxy-2-fluoro analogs of polyprenyl beta-d-arabinofuranosyl phosphates Carbohydr Res 341, 2723–2730 Bosco M, Bisseret P, Constant P & Eustache J (2007) Synthesis of 2¢,3¢-dihydrosolanesyl analogues of b-d-arabinofuranosyl-1-monophosphoryldecaprenol with promising antimycobacterial activity Tetrahedron Lett 48, 153–157 Takayama K, Schnoes HK & Semmler EJ (1973) Characterization of the alkali-stable mannophospholipids of Mycobacterium smegmatis Biochim Biophys Acta 316, 212–221 Wolucka BA & de Hoffmann E (1998) Isolation and characterization of the major form of polyprenyl-phospho-mannose from Mycobacterium smegmatis Glycobiology 8, 955–962 Matsunaga I, Bhatt A, Young DC, Cheng TY, Eyles SJ, Besra GS, Briken V, Porcelli SA, Costello CE, Jacobs WR et al (2004) Mycobacterium tuberculosis pks12 produces a novel polyketide presented by CD1c to T cells J Exp Med 200, 1559–1569 Calderone CT, Kowtoniuk WE, Kelleher NL, Walsh CT & Dorrestein PC (2006) Convergence of isoprene and polyketide biosynthetic machinery: isoprenylS-carrier proteins in the pksX pathway of Bacillus subtilis Proc Natl Acad Sci USA 103, 8977–8982 Crick DC, Schulbach MC, Zink EE, Macchia M, Barontini S, Besra GS & Brennan PJ (2000) Polyprenyl phosphate biosynthesis in Mycobacterium tuberculosis and Mycobacterium smegmatis J Bacteriol 182, 5771– 5778 Gurcha SS, Baulard AR, Kremer L, Locht C, Moody DB, Muhlecker W, Costello CE, Crick DC, Brennan PJ & Besra GS (2002) Ppm1, a novel polyprenol monophosphomannose synthase from Mycobacterium tuberculosis Biochem J 365, 441–450 Baulard AR, Gurcha SS, Engohang-Ndong J, Gouffi K, Locht C & Besra GS (2003) In vivo interaction between the polyprenol phosphate mannose synthase Ppm1 and the integral membrane protein Ppm2 from Mycobacterium smegmatis revealed by a bacterial twohybrid system J Biol Chem 278, 2242–2248 Morita YS, Sena CB, Waller RF, Kurokawa K, Sernee MF, Nakatani F, Haites RE, Billman-Jacobe H, McConville MJ, Maeda Y et al (2006) PimE is a polyprenol-phosphate-mannose-dependent mannosyltransferase that transfers the fifth mannose of phosphatidylinositol mannoside in mycobacteria J Biol Chem 281, 25143–25155 FEBS Journal 275 (2008) 2691–2711 ª 2008 The Author Journal compilation ª 2008 FEBS B A Wolucka 114 Kaur D, Berg S, Dinadayala P, Gicquel B, Chatterjee D, McNeil MR, Vissa VD, Crick DC, Jackson M & Brennan PJ (2006) Biosynthesis of mycobacterial lipoarabinomannan: role of a branching mannosyltransferase Proc Natl Acad Sci USA 103, 13664– 13669 115 Kaur D, McNeil MR, Khoo KH, Chatterjee D, Crick DC, Jackson M & Brennan PJ (2007) New insights into the biosynthesis of mycobacterial lipomannan arising from deletion of a conserved gene J Biol Chem 282, 27133–27140 116 Dinadayala P, Kaur D, Berg S, Amin AG, Vissa VD, Chatterjee D, Brennan PJ & Crick DC (2006) Genetic basis for the synthesis of the immunomodulatory mannose caps of lipoarabinomannan in Mycobacterium tuberculosis J Biol Chem 281, 20027–20035 117 VanderVen BC, Harder JD, Crick DC & Belisle JT (2005) Export-mediated assembly of mycobacterial glycoproteins parallels eukaryotic pathways Science 309, 941–943 118 Mahapatra S, Yagi T, Belisle JT, Espinosa BJ, Hill PJ, McNeil MR, Brennan PJ & Crick DC (2005) Mycobacterial lipid II is composed of a complex mixture of modified muramyl and peptide moieties linked to decaprenyl phosphate J Bacteriol 187, 2747–2757 119 Yagi T, Mahapatra S, Mikusova K, Crick DC & Brennan PJ (2003) Polymerization of mycobacterial arabinogalactan and ligation to peptidoglycan J Biol Chem 278, 26497–26504 120 Besra GS, Sievert T, Lee RE, Slayden RA, Brennan PJ & Takayama K (1994) Identification of the apparent carrier in mycolic acid synthesis Proc Natl Acad Sci USA 91, 12735–12739 121 Moody DB, Ulrichs T, Muhlecker W, Youn DC, Gurcha SS, Grant E, Rosat JP, Brenner MB, Costello CE et al (2000) CD1c-mediated T-cell recognition of isoprenoid glycolipids in Mycobacterium tuberculosis infection Nature 404, 884–888 122 Tanaka Y, Morita CT, Nieves E, Brenner MB & Bloom BR (1995) Natural and synthetic non-peptide antigens recognized by human gamma delta T cells Nature 375, 155–158 123 Zhang Y, Song Y, Yin F, Broderick E, Siegel K, Goddard A, Nieves E, Pasa-Tolic L, Tanaka Y, Wang H et al (2006) Structural studies of Vgamma2Vdelta2 T cell phosphoantigens Chem Biol 13, 985–992 A role for the D-arabinose lipid carrier 124 Wolucka BA, Rozenberg R, de Hoffmann E & Chojnacki T (1996) Desorption chemical ionization tandem mass spectrometry of polyprenyl and dolichyl phosphates J Am Soc Mass Spectrom 7, 958–964 125 Cooks RG, Ouyang Z, Takats Z & Wiseman JM (2006) Detection technologies Ambient mass spectrometry Science 311, 1566–1570 126 Wolucka BA, de Hoffmann E, Rush JS & Waechter CJ (1996) Determination of the anomeric configuration of glycosyl esters of nucleoside pyrophosphates and polyisoprenyl phosphates by fast-atom bombardment tandem mass spectrometry J Am Soc Mass Spectrom 7, 541–549 127 Wolucka BA, Rush JS, Waechter CJ, Shibaev VN & de Hoffmann E (1998) An electrospray-ionization tandem mass spectrometry method for determination of the anomeric configuration of glycosyl 1-phosphate derivatives Anal Biochem 255, 244–251 128 Mulroney B, Barrie Peel J & Traeger JC (1999) Theoretical study of deprotonated glucopyranosyl disaccharide fragmentation J Mass Spectrom 34, 856–871 129 Denekamp C & Sandlers Y (2005) Anomeric distinction and oxonium ion formation in acetylated glycosides J Mass Spectrom 40, 765–771 130 Crich D & Dudkin V (2002) Confirmation of the connectivity of 4,8,12,16,20-pentamethylpentacosylphoshoryl beta-d-mannopyranoside, an unusual beta-mannosyl phosphoisoprenoid from Mycobacterium avium, through synthesis J Am Chem Soc 124, 2263–2266 131 Jensen UG, Brandt NJ, Christensen E, Skovby F, Norgaard-Pedersen B & Simonsen H (2001) Neonatal screening for galactosemia by quantitative analysis of hexose monophosphates using tandem mass spectrometry: a retrospective study Clin Chem 47, 1364–1372 132 Soo EC, Aubry AJ, Logan SM, Guerry P, Kelly JF, Young NM & Thibault P (2004) Selective detection and identification of sugar nucleotides by CE-electrospray-MS and its application to bacterial metabolomics Anal Chem 76, 619–626 133 Lamichhane G, Zignol M, Blades NJ, Geiman DE, Dougherty A, Grosset J, Broman KW & Bishai WR (2003) A postgenomic method for predicting essential genes at subsaturation levels of mutagenesis: application to Mycobacterium tuberculosis Proc Natl Acad Sci USA 100, 7213–7218 FEBS Journal 275 (2008) 2691–2711 ª 2008 The Author Journal compilation ª 2008 FEBS 2711 ... inner linear region of Araf-(1 fi 5) -a- Araf and of branched non-reducing terminal Ara6 motifs: Arafb1 fi 2Arafa1 fi 5(Arafb1 fi 2Arafa1 fi 3)Arafa1 fi 5Arafa1 About two-thirds of the terminal b-Araf and... [3 5–3 7], water-soluble activated forms of d-arabinose, Scheme The original scheme of biosynthesis of D-arabinofuranosyl residues of the cell-wall arabinogalactan and lipoarabinomannan in mycobacteria, ... incorporation of d-arabinofuranosyl residues of decaprenyl-phospho-arabinose into the arabinogalactan and (lipo)arabinomannan of the mycobacterial cell wall at the level of the Emb proteins –