Novel modified version of nonphosphorylated sugar metabolism – an alternative L-rhamnose pathway of Sphingomonas sp. Seiya Watanabe 1,2,3 and Keisuke Makino 1,2,3,4 1 Institute of Advanced Energy, Kyoto University, Japan 2 New Energy and Industrial Technology Development Organization, Gokasho, Uji, Kyoto, Japan 3 CREST, Japan Science and Technology Agency, Gokasho, Uji, Kyoto, Japan 4 Innovative Collaboration Center, Kyoto University, Japan Microorganisms can utilize pentoses and deoxyhexoses as their sole carbon source. There are generally two pathways for the metabolism of these sugars, one with phosphorylated intermediates and the other without such intermediates. The former pathways of bacteria and ⁄ or fungi have been studied extensively. Many Keywords Entner–Doudoroff pathway; gene cluster; L-rhamnose; metabolic evolution; Sphingomonas sp. Correspondence S. Watanabe, Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan Fax: +81 774 38 3524 Tel: +81 774 38 3596 E-mail: irab@iae.kyoto-u.ac.jp (Received 30 October 2008, revised 9 December 2008, accepted 5 January 2009) doi:10.1111/j.1742-4658.2009.06885.x Several bacteria, including Azotobacter vinelandii, possess an alternative pathway of l-rhamnose metabolism, which is different from the known bacterial pathway. In a previous article, a gene cluster related to this path- way was identified, consisting of the genes encoding the four metabolic enzymes l-rhamnose-1-dehydrogenase (LRA1), l-rhamnono-c-lactonase (LRA2), l-rhamnonate dehydratase (LRA3) and l-2-keto-3-deoxyrhamno- nate (l-KDR) aldolase (LRA4), by which l-rhamnose is converted into pyruvate and l-lactaldehyde, through analogous reaction steps to the well- known Entner-Doudoroff (ED) pathway. In this study, bioinformatic analysis revealed that Sphingomonas sp. possesses a gene cluster consisting of LRA1–3 and two genes of unknown function, LRA5 and LRA6. LRA5 catalyzed the NAD + -dependent dehydrogenation of several l-2-keto-3-de- oxyacid-sugars, including l-KDR. Furthermore, the reaction product was converted to pyruvate and l-lactate by LRA6; this is different from the pathway of Azotobacter vinelandii. Therefore, LRA5 and LRA6 were assigned as the novel enzymes l-KDR 4-dehydrogenase and l-2,4-diketo-3- deoxyrhamnonate hydrolase, respectively. Interestingly, both enzymes were phylogenetically similar to l-rhamnose-1-dehydrogenase and d-2-keto-3- deoxyarabinonate dehydratase, respectively, and the latter was involved in the archeal nonphosphorylative d-arabinose pathway, which is partially analogous to the ED pathway. The introduction of LRA1–4 or LRA1–3, LRA5 and LAR6 compensated for the l-rhamnose-defective phenotype of an Escherichia coli mutant. Metabolic evolution and promiscuity between the alternative l-rhamnose pathway and other sugar pathways analogous to the ED pathway are discussed. Abbreviations COG, cluster of orthologous groups of proteins; D-KDA, D-2-keto-3-deoxyarabinonate; D-KGD, D-2-keto-3-deoxygluconate; ED, Entner– Doudoroff; FAH, fumarylacetoacetate hydrolase; L-DKDR, L-2,4-diketo-3-deoxyrhamnonate; L-KDF, L-2-keto-3-deoxyfuconate; L-KDL, L-2-keto- 3-deoxylyxonate; L-KDM, L-2-keto-3-deoxymannonate; L-KDR, L-2-keto-3-deoxyrhamnonate; LRA1, L-rhamnose-1-dehydrogenase; LRA2, L-rhamnono-c-lactonase; LRA3, L-rhamnonate dehydratase; LRA4, L-2-keto-3-deoxyrhamnonate aldolase; LRA5, L-2-keto-3-deoxyrhamnonate- (4)-dehydrogenase; LRA6, L-2,4-diketo-3-deoxyrhamnonate hydrolase; MhpD, 2-oxopent-4-enoate hydratase; npED, nonphosphorylative Entner–Doudoroff; SDR, short-chain dehydrogenase ⁄ reductase; aKGSA, a-ketoglutaric semialdehyde. 1554 FEBS Journal 276 (2009) 1554–1567 ª 2009 The Authors Journal compilation ª 2009 FEBS bacteria, including Escherichia coli, also metabolize l-rhamnose (l-6-deoxymannose) through this type of pathway, using enzymes consisting of l-rhamnose isomerase (EC 5.3.1.14), rhamnulokinase (EC 2.7.1.5), and rhamnulose-1-phosphate aldolase (EC 4.1.2.19) [1]. The l-lactaldehyde obtained, together with dihydroxyacetone phosphate, is further converted to l-lactate or 1,2-propanediol by l-lactaldehyde dehydro- genase (EC 1.2.1.22) [2] and lactaldehyde : propanediol oxidoreductase [EC 1.1.1.77(55)] [3] under aerobic and anaerobic conditions, respectively. The pathways without phosphorylated intermediates are classified into two groups, in which the sugar is commonly converted into a dl-2-keto-3-deoxyacid- sugar through the participation of dehydrogenase, lactonase and dehydratase enzymes (schematic reac- tions a–c in Fig. 1) (each enzyme is referred to as ‘E’ below). In the ‘type I pathway’ of d-glucose [4,5], d-galactose [6], d-fucose [7–10] and l-arabinose [11], the dl-2-keto-3-deoxyacid-sugar is cleaved through an aldolase reaction (schematic reaction e) to the appro- priate aldehyde and pyruvate as well as to the Entner– Doudoroff (ED) pathway. Although most metabolic genes have not yet been identified, except for the ‘non- phosphorylative ED (npED) pathway’ in Archaea, a previous article [12] recently characterized this type of l-rhamnose pathway in the fungi Pichia stipitis and Debaryomyces hansenii (E12–E15) and the bacterium Azotobacter vinelandii (E16–E19). In this pathway, l-rhamnose is converted into pyruvate and l-lactalde- hyde via l-rhamnono-c-lactone and l-rhamnonate, by the consecutive action of the enzymes l-rhamnose- D-Galactose L-Fucose D-Fucose L-Arabinose Pathway Hexaric acids L-Arabinose D-Arabinose II D-Xylose 71 COG2706 COG3386 COG3618 COG2220 COG0129 COG4948 COG0800 COG0329 COG3836 COG0179 COG1012COG0364 COG1063 COG4993 COG0673 COG1028 COG0667 Glyceraldehyde 3P D-Glyceraldehyde L-Lactate D-Lactaldehyde Glycolaldehyde Final product α-Ketoglutarate g 52 55 60 65 70 74 79 84 Hexaric acids L-Rhamnose I Tartronate semialdehyde L-Lactaldehyde L-Lactate Schematic reactions Pathway Domain ED npED Gluconate 5 Final product (pyruvate +) Glyceraldehyde 3P D-Glyceraldehyde – – – 20 D-Galacturonate B E B B B Domain B B B B B A B A B E B B A B B B B E b – – 31 34 38 43 47 57 62 67 76 81 – 13 17 b 2 10 21 – c 32 35 39 44 48 50 53 58 63 68 72 77 82 25 14 18 22 c 3 6 28 f 36 41 45 49 51 54 59 64 69 73 78 83 26 15 19 24 e 4 7 29 – 40 – – – – – – – – – – – – – – 23 d – – – L-Glyceraldehyde a – – 30 33 37 42 46 56 61 66 75 80 – 12 16 a 1 8 9 11 27 III III I I PPP PPP PPP ED Fig. 1. Comparison of sugar metabolic path- ways analogous to the ED pathway in bacte- ria (B), eukaryotes (E), and Archaea (A). Sugar is commonly metabolized through the participation of sugar dehydrogenase (a), lactone-sugar hydrolase (lactonase) (b), acid- sugar dehydratase (c), 2-keto-3-deoxyacid- sugar dehydrogenase (d), aldolase (e), and dehydratase (f) for 2-keto-3-deoxyacid-sugar, and aKGSA dehydrogenase (g). Colored COGs are homologous to each other, and white indicates that the metabolic gene has not yet been identified. Numbers (1–84) correspond to enzymes catalyzing each reaction (listed in Table S1), which are referred to as ‘E’ in the text. In this study, an alternative L-rhamnose pathway including E20–E24 was focused on (indicated by cir- cles). The reactions of E24 and E41 can be assigned as equivalent to e and f (see text). S. Watanabe and K. Makino Novel L-rhamnose metabolic pathway FEBS Journal 276 (2009) 1554–1567 ª 2009 The Authors Journal compilation ª 2009 FEBS 1555 1-dehydrogenase (LRA1; EC 1.1.1.173), l-rhamnono- c-lactonase (LRA2; EC 3.1.1.65), l-rhamnonate dehydratase (LRA3; EC 4.2.1.90), and l-2-keto-3- deoxyrhamnonate (l-KDR) aldolase (LRA4) (referred to as the ‘aldolase pathway’ in this article) (Fig. 2A). Furthermore, the metabolic fate of l-lactaldehyde is dehydrogenation by l-lactaldehyde dehydrogenase (EC 1.2.1.22), which is similar to the known bacterial pathway as described above [13]. Interestingly, there is no evolutionary relationship between the l-KDR aldolases from fungi and bacteria (E15 and E19). l-Lactaldehyde dehydrogenases from fungi and bacte- ria belong to different subfamilies in the aldehyde dehydrogenase superfamily, and show distinct coen- zyme and substrate specificities. These findings indicate that the pathways have evolved independently in spite of the homologous schematic conversion of l-rham- nose. The ‘type II pathway’ without phosphorylated intermediates corresponds to an alternative pathway of d-arabinose [14], l-arabinose [15–17], and d-xylose [18]. In these pathways, the dl-2-keto-3-deoxypento- nate intermediate is converted to a-ketoglutarate via L-Rhamnose:H + symporter EAM07803 EAM07804 EAM07805 EAM07806 EAM07807 EAM07808 EAM07809 EAM07810 Sugar transporterSugar channel Azotobacter vinelandii EAT09360 EAT09361 EAT09362 EAT09364 L-Rhamnose:H + symporter EAT09366 β-Xylosidase EAT09367 EAT09363 EAT09365 Sphingomonas sp. SKA58 (NBRC101715) Pichia stipitis ABN68602 ABN68603ABN68405ABN68404 LRA1 LRA3 LRA6 LRA5 LRA2 LRA1 LRA3LRA2 LRA4 LRA4LRA1 LRA2LRA3 Debaryomyces hansenii CAG87574CAG87575CAG87576CAG87577 LADH Chr 2 ABN64318 LADH CAG90160Chr G LRA4 LRA3 Escherichia coli yfaU yfaV yfaW Transporter Chr8 Chr E L-Rhamnose L-Rhamnono-γ-lactone L-Rhamnonate L-2-Keto-3-deoxyrhamnonate ( L-KDR) L-Rhamnose 1-dehydrogenase ( LRA1, EC 1.1.1.173) L-Rhamnono-γ-lactonase ( LRA2, EC 3.1.1.65) L-Rhamnonate dehydratase ( LRA3, EC 4.2.1.90) NAD(P) + NAD(P)H H2O H2O Pyruvate L-Lactaldehyde L-KDR aldolase ( LRA4) NAD + NADH L-2,4-Diketo-3-deoxyrhamnonate ( L-DKDR) Pyruvate L-Lactate L-DKDR hydrolase ( LRA6) L-KDR 4-dehydrogenase ( LRA5) Aldolase pathway Diketo-hydrolase pathway L-Lactate L-Lactaldehyde dehydrogenase ( LADH, EC 1.2.1.22) NAD + NADH A B Xanthomonas campestris FucDFucB FucCFucA FucE AAM43286 AAM43287 AAM43288 AAM43289 AAM43290 AAM43291 AAM43292 Sugar transporter L-Fucopyranoside mutarotase C Fig. 2. The alternative L-rhamnose pathway (A) and schematic gene clusters (B). P. stipitis, D. hansenii and A. vinelandii possess the ‘aldol- ase pathway’, in which L-rhamnose is converted into pyruvate and L-lactaldehyde. L-Lactaldehyde dehydrogenase then produces L-lactate from L-lactaldehyde in this pathway. In Sphingomonas sp., the L-KDR intermediate is alternatively converted into pyruvate and L-lactate via L-DKDR (diketo-hydrolase pathway). Chr n indicates chromosome number. Homologous genes are indicated in the same color, and corre- spond to Fig. 1. Gray putative genes are similar in sequence to other L-rhamnose-related enzymes involved in sugar uptake. (C) Schematic gene cluster related to the alternative L-fucose pathway of bacteria [33]. Novel L-rhamnose metabolic pathway S. Watanabe and K. Makino 1556 FEBS Journal 276 (2009) 1554–1567 ª 2009 The Authors Journal compilation ª 2009 FEBS a-ketoglutaric semialdehyde (aKGSA) by dl-2-keto-3- deoxypentonate dehydratase (EC 4.1.2.18; E59, E64, E69, E73, E78, and E83) and aKGSA dehydrogenase (EC 1.2.1.26; E60, E65, E70, E74, E79, and E80) (sche- matic reactions e and f, respectively, in Fig. 1). aKGSA is also produced from hexaric acids (d-gluca- rate and d-galactarate) via d-2-keto-3-deoxyglucarate by two successive dehydration reactions in bacteria (E50–E52 and E53–E55) [19]. Most of these alternative pathways of sugar metabo- lism have been described on the basis only of enzyme activities in cell-free extracts from experiments performed several decades ago. It could be useful to identify a set of the metabolic genes (enzymes) for the enzymatic synthesis of unavailable specific intermedi- ates, in particular dl-2-keto-3-deoxyacid-sugars. In this regard, transcriptomic and ⁄ or proteomic analysis have significant advantages, as shown in the alternative d-arabinose pathway of Archaea [14]. We, alternatively, focused on the following two insights: (a) the equivalent reaction step-catalyzing metabolic enzymes involved in sugar pathways analogous to the ED pathway are classi- fied into limited numbers of the known protein families, cluster of orthologous groups of proteins (COG) (Fig. 1); (b) metabolic genes often form a single gene cluster in the genomes of bacteria and Archaea (Fig. 2B). Accordingly, homology searches were carried out using the known metabolic genes (Table S1) against the genomes of microorganisms, and ‘semi-automati- cally’ selected a set of four potential metabolic genes, LRA1–4, involved in the alternative l-rhamnose path- way described above [12], suggesting that this approach may be helpful in identifying unknown sugar pathways analogous to the ED pathway, even if the phenotype of the microorganism is not available. This study further developed this possibility and revealed that in Sphingomonas sp., a phenotype- unknown but genome sequence-available bacterium, l-rhamnose is converted into pyruvate and l-lactate (but not l-lactaldehyde) via four nonphosphorylated intermediates by five metabolic enzymes (genes), which differed partially from the aldolase pathway. Compari- sons between the novel l-rhamnose pathway and other sugar metabolic pathways and the substrate promiscuity in the metabolic enzymes are also described. Results Gene cluster related to L-rhamnose metabolism in Sphingomonas sp. Several significant insights were obtained about sugar pathways analogous to the ED pathway, including the alternative l-rhamnose pathways of fungi and bacteria (Fig. 1). Therefore, an extended bioinformatic analysis was carried out, and an interesting gene cluster related to putative sugar metabolism was found in the genome of Sphingomonas sp. SKA58 (Fig. 2B). In this article, the prefixes Ps (P. stipitis), Dh (D. hansenii), Av (A. vinelandii) and Sp (Sphingomonas sp.) have been added to gene symbols or protein designations when required for clarity. When compared with the LRA1–4 gene clusters of fungi and bacteria, the gene EAT09362 of Sphingo- monas sp. was homologous to the LRA3 gene encod- ing l-rhamnonate dehydratase (COG4948, enolase superfamily); there was 80.2% identity with AvLRA3. Furthermore, gene EAT09365 belonged to the same COG group as the LRA2 gene encoding l-rhamnono- c-lactonase (COG3618, a ⁄ b hydrolase fold enzymes). On the other hand, the gene cluster of Sphingomonas sp. SKA58 contained the genes encoding two putative short-chain dehydrogenase ⁄ reductase (SDR) family enzymes (COG1028), EAT09360 (SpLRA1) and EAT09364 (SpLRA5) (Fig. 2B). As the former showed higher amino acid sequence homology to other LRA1 proteins than the latter (66.7% and 25.3% identity with AvLRA1, respectively), the enzyme function of EAT09360 is likely to play a role as an l-rhamnose-1-dehydrogenase (see below). On the other hand, there was no homolog to the EAT09363 gene in the LRA1–4 gene cluster: COG0179, 2-oxopent-4-enoate hydratase (MhpD) family. These results indicated that the gene cluster of Sphingomonas sp. SKA58 may be responsible for non- phosphorylative sugar metabolism, in which the sche- matic conversion of the l-2-keto-3-deoxyacid-sugar intermediate is different from the aldol cleavage by l-KDR aldolase. In this study, we used Sphingomonas sp. NBRC 101715 instead of Sphingomonas sp. SKA58 as a target microorganism; between these, there is 99.8% identity in 16S rRNA sequence. We were successful in amplifying SpLRA genes by geno- mic PCR using oligonucleotide primers designed from the Sphingomonas sp. SKA58 genome sequence (see below). Unless otherwise noted, Sphingomonas sp. hereafter indicates strain NBRC 101715. Functional expression of SpLRA genes in E. coli Among the five SpLRA gene products, SpLRA3, SpLRA5, and SpLRA6 were successfully expressed in E. coli cells as (His)6-tagged enzymes. The recombi- nant enzymes were purified to homogeneity using a nickel-chelating affinity column and then gel filtration chromatography (Fig. 3B). Western blot analysis with S. Watanabe and K. Makino Novel L-rhamnose metabolic pathway FEBS Journal 276 (2009) 1554–1567 ª 2009 The Authors Journal compilation ª 2009 FEBS 1557 an antibody against (His)6-tag confirmed the (His)6- tag at the N-terminal. L-Rhamnose-1-dehydrogenase (SpLRA1) To initially estimate the physiological role of the LRA gene cluster, we first attempted to biochemically char- acterize a dehydrogenase with l-rhamnose in Sphingo- monas sp. When compared with nutrient medium (0.035 unitÆmg )1 protein), approximate 9.7-fold higher activity of NADP + -dependent dehydrogenation with l-rhamnose was found in the cell-free extract from Sphingomonas sp. cells grown on l-rhamnose (0.34 uni- tÆmg )1 protein). Similar induction of NAD + -dependent activity by l-rhamnose was also found, although the specific activity was slightly lower than the NADP + - dependent activity. The l-rhamnose-1-dehydrogenase was (partially) purified by four chromatographic steps: there was NADP + -dependent and NAD + -dependent specific activity of 39.8 and 31.7 unitÆmg )1 protein, respectively (Fig. 3A). A typical result of purification is summarized in Table 1A. During the purification procedure, the ratio of NADP + -linked and NAD + - linked activity remained almost constant, suggesting the presence of only one protein as l-rhamnose-1- dehydrogenase. Two major protein bands were found on SDS ⁄ PAGE gel (bands A and B, respectively, in Fig. 3A). The N-terminal amino acid sequence up to 20 amino acids of band B, MKLLEGKTVLITGAST GIGR, was completely identical to that of SpLRA1, and the putative molecular mass of SpLRA1 (26.5 kDa) was similar to that of the native enzyme ( 28 kDa) (band A is described in the next section). Significant dehydrogenase activity was observed with l-rhamnose, l-lyxose (75%) and l-mannose (8.4%) in the presence of NADP + [values in parentheses are activity relative to that with l-rhamnose (100%)]. The substrate specificity and dual coenzyme specificity between NAD + and NADP + (see above) were similar to the same bacterial AvLRA1 (high concomitant activity for l-lyxose), compared with fungal PsLRA1 and DhLRA1 (strict NAD + -dependence) [12]. These results indicated that SpLRA1 encodes NAD(P) + - dependent l-rhamnose-1-dehydrogenase, and that the 91 65 48 37 28 M1 2 3kDakDa 195 119 91 65 48 37 28 12 34MM5 AB 20.5 Band A Band B Fig. 3. SDS ⁄ PAGE. (A) Purification of native L-rhamnose dehydro- genase from Sphingomonas sp. Lane 1: cell-free extract (100 lg). Lane 2: HiPrep 16 ⁄ 10 Q FF (50 lg). Lane 3: HiPrep 16 ⁄ 10 Butyl FF (20 lg). Lane 4: hydroxyapatite (20 lg). Lane 5: HiLoad 26 ⁄ 60 Superdex 200 pg (20 lg). M is marker protein. Bands A and B correspond to L-KDR 4-dehydrogenase and L-rhamnose-1-dehydro- genase, respectively (see text). (B) Purification of (His)6-tagged SpLRA3 (lane 1), SpLRA5 (lane 2), and SpLRA6 (lane 3) (each of 5 lg). Table 1. Summary of concomitant purification of L-rhamnose-1-dehydrogenase (A) and L-KDR 4-dehydrogenase (B) from Sphingomonas sp. Step Total protein (mg) Total activity (units) Specific activity a (unitsÆmg )1 protein) Yield (%) Purification fold NAD + NADP + NADP + ⁄ NAD + A Cell-free extract 1325 654 821 1.26 0.620 100 1.0 HiPrep 16 ⁄ 10 Q FF 212 544 755 1.39 3.56 92 1.0 HiPrep 16 ⁄ 10 Butyl FF 28.8 437 535 1.22 18.6 65 4.4 CHT ceramic hydroxyapatite 13.8 202 274 1.36 19.9 33 12 HiLoad 26 ⁄ 60 Superdex 200 pg 3.72 118 148 1.25 39.8 18 12 Step Total protein (mg) Total activity (units) Specific activity (unitsÆmg )1 protein) Yield (%) Purification fold B Cell-free extract 1325 263 0.198 100 1.0 HiPrep 16 ⁄ 10 Q FF 212 118 0.557 45 2.8 HiPrep 16 ⁄ 10 Butyl FF 28.8 49.2 1.71 19 8.6 CHT ceramic hydroxyapatite 13.8 30.5 2.21 12 11 HiLoad 26 ⁄ 60 Superdex 200 pg 3.72 20.4 5.48 8 28 a NADP + -dependent activity. Novel L-rhamnose metabolic pathway S. Watanabe and K. Makino 1558 FEBS Journal 276 (2009) 1554–1567 ª 2009 The Authors Journal compilation ª 2009 FEBS remaining LRA genes may also be related to l-rham- nose metabolism. L-2-Keto-3-deoxyrhamnonate-(4)-dehydrogenase (SpLRA5) As described above, although SpLRA5 belongs to the SDR family, together with SpLRA1, the enzyme func- tion may be different from the dehydrogenation with l-rhamnose. At present, this protein family contains approximately 3000 primary structures and consists of enzymes of several EC classes [20], and relatively high degrees of similarity to SpLRA5 were found in several (putative) reductases ⁄ dehydrogenases (30–35% identi- ties). Significant NAD + -dependent dehydrogenation activity (0.22 unitÆmg )1 protein) of l-KDR was found when Sphingomonas sp. was grown on l-rhamnose, but not when it was grown on nutrient medium. Further- more, no aldolase activity of l-KDR was induced by l-rhamnose. Surprisingly, the dehydrogenase for l-KDR was concomitantly purified with l-rhamnose- 1-dehydrogenase: NAD + -dependent specific activity of 5.48 unitÆmg )1 protein was found (Table 1B). In the (partially) purified sample, band A should correspond to this enzyme (Fig. 3A), and the N-terminal amino acid sequence up to 19 amino acids was significantly homologous with that of SpLRA5: (M) SVFAGRYA GRXAIVTGGAS (underlined letters indicate the same amino acids as SpLRA5; X, residue was not deter- mined). On the other hand, the molecular mass of the native enzyme estimated from SDS ⁄ PAGE ( 29 kDa) was slightly higher than the value estimated from the putative amino acid sequence of SpLRA5 (25.7 kDa), although this is not entirely clear. These results suggested that SpLRA5 plays a role as a novel NAD + -dependent l-KDR dehydrogenase involved in l-rhamnose metabolism in Sphingomonas sp. When the purified (His)6-tagged SpLRA5 was incu- bated with each l-2-keto-3-deoxyacid-sugar in the presence of NAD + , clear dehydrogenation activity was detected by a spectrophotometric assay, and the determined kinetic parameters are shown in Table 2. The k cat ⁄ K m value with l-KDR was 81 min )1 Æmm )1 in the range expected for the physiological substrate; this was 9.3-fold and 214-fold higher than those with l-2-keto-3-deoxylyxonate (l-KDL) and l-2-keto- 3-deoxymannonate (l-KDM), respectively, this being mainly caused by the higher value of k cat . l-Rhamnose was an inactive substrate, and no activity was found in the presence of NADP + when l-KDR was used as a substrate. To identify the hydrogen absorbed by SpLRA5, an attempt was made to isolate the reaction product free of NAD + , protein, and buffer, but this proved to be unsuccessful, probably because of the unstable nature of the product. Therefore, the reaction product of SpLRA5 was esti- mated by a coupling reaction with SpLRA6, as described below. The common active center of SDR enzymes consists of a tightly conserved Ser-Tyr-Lys catalytic triad [20]. Furthermore, the coenzyme-binding mode follows a classical ‘Rossmann fold’, in which a characteristic GXXXGX[G ⁄ A] fingerprint motif exists. These motifs are also conserved in SpLRA5: Ser143–Tyr156–Lys160 and Gly17-Gly-Ala-Ser-Gly-Leu-Gly23 (Fig. 4A). These findings suggested that the fundamental catalytic mechanism and coenzyme recognition of SpLRA5 may be similar to those in known SDR enzymes. The recombinant SpLRA5 enzyme formed a homotetra- meric structure by itself, like other SDR proteins (data not shown). Therefore, it is likely that SpLRA1 is completely unnecessary to maintain the active form of SpLRA5, and that concomitant purification of SpLRA5 and SpLRA1 (Fig. 3A) is due to the similar properties on the surface rather than the hetero-oligo- meric structure, although their sequence identity is only 29%. L-2,4-Diketo-3-deoxyrhamnonate hydrolase (SpLRA6) SpLRA6 is a novel member of the MhpD family (COG0179), which is different from the protein fami- lies of LRA1–4 proteins (Fig. 1). In HPLC analysis (Fig. 5A), the retention time of the reaction product of SpLRA5 was almost the same as that of l-KDR Table 2. Kinetic parameters of recombinant SpLRA5 protein. Values are the means ± standard deviation, n =3. Substrate Specific activity a (unitsÆmg )1 protein) K m b (mM) k cat b (min )1 ) k cat ⁄ K m b (min )1 ÆmM )1 ) L-KDR 1.95 ± 0.04 0.646 ± 0.086 51.8 ± 2.6 81.0 ± 6.3 L-KDL 0.697 ± 0.025 1.27 ± 0.11 11.0 ± 0.4 8.72 ± 0.42 L-KDM 0.024 ± 0.001 1.90 ± 0.06 0.720 ± 0.024 0.378 ± 0.001 a Under standard assay conditions as described in Experimental procedures. b Ten different concentrations of substrate between 0.2 and 10 m M were used. S. Watanabe and K. Makino Novel L-rhamnose metabolic pathway FEBS Journal 276 (2009) 1554–1567 ª 2009 The Authors Journal compilation ª 2009 FEBS 1559 ( 10 min). Similar results were also observed with l-KDL and l-KDM. On the other hand, when l-KDR was incubated with SpLRA5 and SpLRA6 in the presence of NAD + , a novel peak with a later retention time (13.1 min) appeared, identical to that of l-lactate. In the case of l-KDL, the peak corresponded to glycolate. Clearer results were obtained with l-KDM: two peaks that differed from that of l-KDM ( 10 and  11.4 min) were observed, and were found to be identical to those of pyruvate and (dl-)glycerate, respectively. These results indicated that SpLRA6 cata- lyzes the hydrolysis of l-2,4-diketo-3-deoxyacid-sugar C O O H H H H O H H O H C H 3 O L-KDR L-KDF C O O H H H H O H H O H C H 3 O C O O H H O H H O H H O H H O H H H O H 1 2 3 4 5 6 D-Gluconate EC 1.1.1.215 D-2-Deoxygluconate C O O H H H H O H H O H H O H H H O H C O O H H H H O H H O H H H O H O D-KDG EC 1.1.1.126 EC 1.1.1.127 (KduD) L-Rhamnose C O O H H O H H O H H O H H O H H H O H C O O H H H H O H H O H H H O H O H O H O H H O H H O H H 3 C H O EC 1.1.1.264 (IdnD) C O O H H O H H O H H O H H H H O H H O L-Idonate C O O H H H H O O H O H O H C H 3 C O O H H H O H O H C H 3 O H 2 O L-DKDR hydrolase (EC 3.7.1 ) Pyruvate L-Lactate L-DKDR C O O H H H O H H O H C O O H H C O O H H H O H H H C O O H H C O O H H H 2 O FAH (EC 3.7.1.2) 4-Fumarylacetoacetate Fumarate Acetoacetate C O O H O H H H H C O O H H H C O O H O H C O O H H H C O O H H H CO 2 HpcE (EC 4.1.1.68) 5-Oxopent-3-ene- 1,2,5-tricarboxylate 2-Oxohept-3- ene-1,7-dioate C O O H H H C H 2 O H H O H O C O O H H H C H O H H O H 2 O D-KDA dehydratase (EC 4.2.1 ) D-KDA αKGSA C H 2 H H H O C O O H H O H H O C O O H C H 3 H 2 O MhpD (EC 4.2.1.80) 4-Hydroxy-2- oxopentanoate 2-Hydroxy-2,4- pentadienoate EC 1.1.1.69 (IdnO/Gno) EC 1.1.1.125 (KduD) EC 1.1.1.173 D W E V E L G D W E V E L G L P E P E L A H Y E A E L V D M E L E M A R I E A E I A N D V S E R F N Q N D L S E R E F Q D D V S A R D L E N D Y A I R D Y L N D W S A R D I Q L E V V G S - R I p L R A 6 u c E d a D S F K H p c E A H F M h p D W 1 1 7 K W 1 2 2 K L 1 4 1 K N 2 7 4 K P 1 9 7 K T 1 0 3 N W 1 4 9 K W 1 5 4 K L 1 6 3 K N 3 0 6 K P 2 3 2 K T 1 3 7 N K Q - - - L E - - - A E N - - E N - - - Q W E Y V R D W S I W S K G K G H D T W V K G K S A D T L P Q S K I Y A G N L R V K S R D G P F L G K S F - - T V A D N A S C G P G D L M I T G T P P G V G P G D V I S T G T P P G V G D G T I L T T G T A I V P G P G D M I A T G T P K G L S P G D L L A S G T I S G S D T G D I I L T G A L G P M V W 2 3 0 K W 2 3 5 K L 2 4 8 K N 3 8 7 K P 3 4 2 K T 2 2 4 N R G T Q H G G Q - P L Y Y Y R P - P L G Q F V D     A v L R A 1 S p L R A 1 S p L R A 5 F u c D I d n O K d u D V S S I V S S I L A S V S A L V G G A M Q T H Y T P T K A G S A L V G G E Y Q T H Y T P T K A G A G K E G N P N A S A Y S A S K A G M S S V A S S I K G V P N R F V Y G V T K A A I C S V Q I A S M L S S E L A R P G I A P Y T A T K G A F Q G G I P V P S Y T A S K K R - - - - - G G 1 4 4 A Q 1 4 2 A D 1 4 1 V D 1 4 1 T L 1 4 5 T R 1 4 3 S V T G A S R G I G R A V T G A S T G I G R A V T G G A S G L G K Q I T A A G A G I G R E V T G A S R G I G L T I T G C D T G L G Q G 0 1 1 1 5 2 0 1 6 1 5 1 1 A B C D * * * * * * * Fig. 4. (A) Partial sequence alignment between L-rhamnose-1-dehydrogenases from A. vinelandii (AvLRA1) and Sphingomonas sp. (SpLRA1, this study), L-KDR 4-dehydrogenase from Sphingomonas sp. (SpLRA5, this study), L-KDF 4-dehydrogenase from X. campestris (FucD), D-glu- conate 5-dehydrogenase from Gluconobacter oxydans (IdnO, CAA56322), and D-KDG 5-dehydrogenase from Erwinia chrysanthemi (KduD, CAA43989). Open and closed circles indicate NAD(H) + -dependent and NADP(H) + -dependent enzymes, respectively. Asterisks, GXXXGX(G ⁄ A) coenzyme-binding motif of Rossmann fold; diamond, Ser-Tyr-Lys catalytic triad. (B) Structurally analogous nonphosphorylated substrate to L-KDR. Each dehydrogenase acts at the gray-shadowed boxes. Enzymes in boxes belong to the SDR protein family as described in (A). (C) Partial sequence alignment between L-DKDR hydrolases from Sphingomonas sp. (SpLRA6, this study) and X. campestris (FucE), D-KDA dehydratase from S. solfataricus (KdaD; Protein Data Bank ID 2Q18), HpcE from E. coli (1I7O), FAH from mouse (1QQJ), and MhpD from E. coli (1SV6). Circles, metal ion ligands; triangles, active sites. The full (structure-based) sequence alignment is shown in Fig. S2. (D) Schematic reactions of MhpD family enzymes. Black triangles indicate cleavage sites of C–C bonds. Novel L-rhamnose metabolic pathway S. Watanabe and K. Makino 1560 FEBS Journal 276 (2009) 1554–1567 ª 2009 The Authors Journal compilation ª 2009 FEBS to pyruvate and hydroxyl acid [physiologically, l-2,4-diketo-3-deoxyrhamnonate (l-DKDR) hydrolase] and that SpLRA5 should be assigned as ‘l-KDR 4-dehydrogenase’, which produces l-DKDR from l-KDR. The MhpD family contains the archetypal MhpD (EC 4.2.1.80) [21] fumarylacetoacetate hydrolase (FAH; EC 3.7.1.2) [22], 5-oxopent-3-ene-1,2,5-tricarboxy- late decarboxylase ⁄ 2-hydroxyhepta-2,4-diene-1,7-dioate isomerase (EC 4.1.1.68) [23], and d-2-keto-3-deoxyara- binonate (d-KDA) dehydratase [14,24] (Fig. 4C; the full sequence alignment is shown in Fig. S2). Among them, FAH catalyzes the hydrolytic cleavage of a C–C bond in fumarylacetoacetate to yield fumarate and acetoacetate, and the catalytic reaction is partially analogous to that of l-DKDR hydrolase (Fig. 4D). Furthermore, it is noteworthy that d-KDA dehydratase is involved in an archeal d-arabinose pathway that is (partially) analogous to the npED pathway and similar to the alter- native l-rhamnose pathway (E73) (Fig. 1). MhpD family enzymes contain Ca 2+ (5-oxopent-3-ene-1,2,5- tricarboxylate decarboxylase ⁄ 2-hydroxyhepta-2,4-diene- 1,7-dioate isomerase and FAH) or Mg 2+ (d-KDA dehydratase) in the active center; these are coordinated with two highly conserved glutamates and one aspartate (Fig. 4C). l-DKDR hydrolase also possesses a structur- ally equivalent metal ion-binding site, Glu119–Glu121– Asp150, whereas there are several significant variations C O O H H H H O H H O H C H 3 O C O O H H H O H O H C H 3 O C O O H H O H C H 3 + NAD + NADH H2O L-Lactate C O O H C H 3 O L-KDR Pyruvate L-2,4-Diketo- 3-deoxyrhamnonate C O O H O H H H O H C H 2 O H C O O H C + Glyocolate NAD + NADH H2O L-KDL C O O H C H 3 O H 2 O H L-2,4-Diketo- 3-deoxylyxonate C O O H H H C H 2 O H O O C O O H H H H O H H O H C H 2 O H O C O O H H H O H O H C H 2 O H O C O O H H O H C H 2 O H C O O H C H 3 O + Glycerate NAD + NADH H2O L-KDM L-2,4-Diketo- 3-deox y mannonate 0 200 0 200 0 200 0 200 400 0 200 0 200 0 200 400 8 9 10 11 12 13 14 0 200 0 200 0 200 400 8 9 10 11 12 13 14 L-KDR + SpLRA5 + NAD + L-KDR + SpLRA5 + NAD + + SpLRA6 L-KDR L-KDL + SpLRA5 + NAD + L-KDL + SpLRA5 + NAD + + SpLRA6 L-KDM + SpLRA5 + NAD + L-KDM + SpLRA5 + NAD + + SpLRA6 0 200 Pyruvate + Glycolate 8 9 10 11 12 13 14 Pyruvate + L-Lactate Retention time (min) mV mV mV Retention time (min) Retention time (min) mV L-KDL L-KDM 0 200 Pyruvate + DL-Glycerate A B Fig. 5. (A) HPLC analysis of the reaction products from L-KDR, L-KDL and L-KDM formed by SpLRA5 and SpLRA6. Authentic pyruvate, L-lac- tate, glycolate and DL-glycerate were present at a concentration of 10 mM. Arrows indicate the peak corresponding to pyruvate produced from L-KDM. (B) Schematic of enzyme reaction products from L-KDR, L-KDL and L-KDM formed by L-KDR 4-dehydrogenase (LRA5) and L-DKDR hydrolase (LRA6). Pyruvate is shown in the dashed box. S. Watanabe and K. Makino Novel L-rhamnose metabolic pathway FEBS Journal 276 (2009) 1554–1567 ª 2009 The Authors Journal compilation ª 2009 FEBS 1561 in several amino acids in the (putative) active sites, which may reflect their different catalytic reactions. L-Rhamnonate dehydratase (SpLRA3) As AvLRA3 utilizes only l-rhamnonate, l-lyxonate and l-mannonate as substrates efficiently [13], other (dl-)2-keto-3-deoxyacid-sugars (and 2-diketo-3-deoxy- acid-sugars) in addition to l-KDR, l-KDL and l-KDM are unavailable as substrates for SpLRA5 and SpLRA6. Therefore, there is still a possibility that the gene cluster of Sphingomonas sp. is responsible for the metabolism not only of l-rhamnose but also of other sugars. In this regard, SpLRA3 is more suitable for estimating the physiological role of the gene cluster as well as of SpLRA1, because a library was constructed previously of 11 acid-sugars as potential substrates for acid sugar dehydratases [12]. Therefore, recombinant (His)6-tagged SpLRA3 was prepared using the same procedures as for SpLRA5 and SpLRA6 (Fig. 3B). By semicarbazide endpoint measurement, significant activity of SpLRA3 was found with l-rhamnonate, l-lyxonate (128%), and l-mannonate (89.2%) [values in parentheses are rela- tive to the activity with l-rhamnonate (100%)]. Furthermore, SpLRA3 showed similar kinetic para- meters with l-rhamnonate to those of AvLRA3 [12] (Table 3), and the k cat ⁄ K m value with l-rhamnonate (177 min )1 Æmm )1 ) was 26.2- and 59.2-fold higher than those with l-lyxonate (6.75 min )1 Æmm )1 ) and l-manno- nate (2.99 min )1 Æmm )1 ), respectively, mainly due to significantly higher K m values (13.6- and 30.2-fold, respectively). These results clearly suggested that SpLRA3 should be assigned as an ‘l-rhamnonate dehydratase’ and that the gene cluster of Sphingomonas sp. is related only to l-rhamnose metabolism. In vivo expression of LRA genes in an L-rhamnose-defective E. coli mutant We here identified an alternative l-rhamnose pathway in Sphingomonas sp. (referred to as the ‘diketo-hydro- lase pathway’) that differs from the complete analo- gous pathway to npED pathway (aldolase pathway) (Fig. 2A). To estimate the physiological meaning of both pathways of l-rhamnose metabolism in vivo, genes LRA1–6 were introduced into plasmid vectors for multiple gene expression and transformed into an l-rhamnose-defective E. coli mutant, the KRX strain (Fig. S1 and Table S1). In this study, the genes for AvLRA1, DhLRA2, AvLRA3, PsLRA4, AvLRA4, SpLRA5 and SpLRA6 were used, because of expres- sion level and ⁄ or solubility problems with other LRA proteins in E. coli cells (see Experimental procedures). Western blot analysis using (His)6-tag attached to the N-terminus of all LRA proteins revealed their func- tional expression in E. coli cells grown on a nutrient medium supplemented with 0.2% (w ⁄ v) l-rhamnose (Fig. 6A). On the other hand, when the recombinant E. coli strains were cultivated in a minimal medium containing 2% (w ⁄ v) l-rhamnose, the l-rhamnose-neg- ative phenotype was compensated for by introduction of the genes LRA1–4 [Duet-1234(Ps) and Duet- 1234(Av)] or LRA1–3, LRA5, and LRA6 (Duet-12356) (Fig. 6B), suggesting significant physiological roles of both of the alternative l-rhamnose pathways in vivo. Unexpectedly, the introduction of only LRA1 and LRA2 also led to slow growth of the cells (see Duet- 12). Indeed, E. coli possesses genes homologous to LRA3 and LRA4 (that encoding yfaW, 63.4% identity with AvLRA3; that encoding yfaU, 50.2% identity with AvLRA4) (Fig. 2B), and their enzyme functions were recently assigned as l-rhamnonate dehydratase and l-KDR aldolase, respectively [25,26]. Discussion As illustrated in Fig. 1, there is significant phylogenetic mosaicism between the metabolic enzymes involved in sugar pathways analogous to the ED pathway: sugar dehydrogenase, lactone-sugar hydrolase (lactonase), acid-sugar dehydratase, and aldolase and dehydratase for (dl-)2-keto-3-deoxyacid-sugar. One of the most interesting findings in this study was that two enzymes belonging to the same protein family (COG1028) are involved in a single sugar metabolic pathway: LRA1 Table 3. Kinetic parameters of recombinant SpLRA3 protein. Values are the means ± standard deviation, n =3. Substrate Specific activity a (unitsÆmg )1 protein) K m (mM) k cat (min )1 ) k cat ⁄ K m (min )1 ÆmM )1 ) L-Rhamnonate b 0.401 ± 0.03 (0.891 ± 0.058) c 0.121 ± 0.006 (0.115 ± 0.001) 21.3 ± 0.6 (43.1 ± 0.2) 177 ± 5 (375 ± 3) L-Lyxonate c 0.196 ± 0.02 1.64 ± 0.11 11.0 ± 0.5 6.75 ± 0.14 L-Mannonate d 0.134 ± 0.01 3.66 ± 0.67 10.8 ± 1.4 2.99 ± 0.16 a Under standard assay conditions as described in Experimental procedures. b Ten different concentrations of substrate between 0.02 and 1m M were used. c Values of AvLRA3 [12]. d Six different concentrations of substrate between 0.5 and 5 mM were used. Novel L-rhamnose metabolic pathway S. Watanabe and K. Makino 1562 FEBS Journal 276 (2009) 1554–1567 ª 2009 The Authors Journal compilation ª 2009 FEBS and LRA5. In the ‘recruitment model’ of enzyme evo- lution proposed by Jensen [27], new enzymes evolve by duplication and mutation of the same enzyme classes from other pathways, leading to ‘catalytically promis- cuous’ enzymes and ‘patchwork’-like metabolic path- ways. In case of the diketo-hydrolase pathway of Sphingomonas sp., similar evolutionary processes have occurred more than once in the same pathway. The ED pathway often plays a role as the metabolic funnel of the npED pathway. For example, in several hyperthermophilic Archaea, d-2-keto-3-deoxygluconate (d-KDG) and glycerate (produced from d-glyceralde- hyde) are phosphorylated by specific kinases and subsequently metabolized through the ED pathway: the so-called ‘semi-phosphorylative ED pathway’ [28]. On the other hand, in several bacterial pathways, d-gluconate and ⁄ or d-KDG is produced from d-2,5-di- keto-3-deoxygluconate (in pectin degradation [29]), d-5- dehydrogluconate (in the l-idonate pathway [30]), and d-2-deoxygluconate (in the 2-deoxyglucose pathway [31]), and the (reverse) reactions, in particular the first reaction, are analogous to that of SpLRA5. Therefore, it may be reasonable to suppose that l-KDR 4-dehydro- genase is phylogenetically similar to the enzymes cata- lyzing these reactions [29,32] (Fig. 4A,B). To the best of our knowledge, the conversion of l-KDR into pyruvate and l-lactate via l-DKDR is a novel metabolic fate in the known sugar pathways. Gerlt et al. [33] recently identified a gene cluster related to the alternative l-fucose metabolism of the bacterium Xanthomonas campestris (E37–E41) (Fig. 2B). Among the protein products of the five metabolic genes, FucB, FucC, FucD, and FucE are homologous to SpLRA2 (28% identity), SpLRA3 (30% identity), SpLRA5 (35% identity; see Fig. 4A), and SpLRA6 (56% identity, see Fig. 4D), respectively, suggesting that l-DKDR is also produced from l-2-keto-3-deoxyfuco- nate (l-KDF) in this pathway. On the other hand, as l-fucose and l-fuconate are inactive substrates for SpLRA1 and SpLRA3, respectively (see Results), the LRA gene cluster should be related only to l-rhamnose metabolism of Sphingomonas sp. but not l-fucose metabolism; in contrast, the archaeon Sulfolobus solfataricus metabolizes both d-glucose and d-galactose promiscuously through the npED pathway [4,5]. l-Lactaldehyde is subsequently converted to l-lactate by l-lactaldehyde dehydrogenase in the aldolase path- way [13], whereas the continuous reactions of l-KDR 4-dehydrogenase and l-DKDR hydrolase allow the metabolism of l-rhamnose into the same pyruvate and l-lactate products without involvement of the physio- logically toxic aldehyde (Fig. 2A), by which the diketo- hydrolase pathway may be more favorable than the aldolase pathway. Another interesting insight obtained in this study is that l-DKDR hydrolase belongs to the same MhpD protein family as d-KDA dehydratase (Fig. 4C). As mentioned previously, there is another version of the npED pathway (type II in Fig. 1), in which the dl-2-keto-3-pentonate intermediate is converted to 50 30 0 1 12 123 1235 12356 M 1 1 2 1 2 3 1 2 3 4 1 3 4 2 1 3 5 2 3 1 5 2 6 1234(Ps) 1234(Av) kDa E. coli Duet strains A B 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 2 4 6 8 10 12 A600 Da y s 1234(Av) 1234(Ps) 12356 12 0 Fig. 6. Expression of LRA genes in an L-rhamnose-defective mutant of E. coli. The constructed recombinant E. coli strains are summarized in Table S3. (A) Western blot analysis. All LRA enzymes were overexpres- sed as (His)6-tagged proteins in E. coli cells grown in a nutrient medium supplemented with L-rhamnose, and purified on an Ni 2+ -chelating affinity column. One hundred micrograms of each of the purified proteins was applied to 11% (w ⁄ v) gel. M indicates marker protein. (B) Growth in M9 minimal liquid medium supplemented with 2% (w ⁄ v) L-rhamnose. S. Watanabe and K. Makino Novel L-rhamnose metabolic pathway FEBS Journal 276 (2009) 1554–1567 ª 2009 The Authors Journal compilation ª 2009 FEBS 1563 [...]... (2008) Eukaryotic and bacterial gene clusters related to an alternative pathway of non-phosphorylated L-rhamnose metabolism (2008) J Biol Chem 283, 2037 2–2 0382 Watanabe S, Piyanart S & Makino K (2008) Metabolic fate of L-lactaldehyde derived from an alternative L-rhamnose pathway FEBS J 275, 513 9–5 149 Brouns SJ, Walther J, Snijders AP, van de Werken HJ, Willemen HL, Worm P, de Vos MG, Andersson A, Lundgren... insight into an alternative pathway of bacterial L-arabinose metabolism J Biol Chem 281, 2887 6– 28888 17 Watanabe S, Shimada N, Tajima K, Kodaki T & Makino K (2006) Identification and characterization of L-arabonate dehydratase, L-2-keto-3-deoxyarabonate dehydratase and L-arabinolactonase involved in an alternative pathway of L-arabinose metabolism: novel evolutionary insight into sugar metabolism J... bacterial and archeal genome sequences using the metabolic genes involved in an alternative l-rhamnose pathway of P stipitis, D hansenii and A vinelandii LRA 1– 4 Candidate genes showing significant homology were further examined by estimating whether these enzymes belong to the same protein family as the probe proteins in the flanking region Functional expression and purification of (His)6-tagged proteins Sphingomonas. .. of Japan (to S Watanabe), the Fermentation and Metabolism Research Foundation, the Japan Bioindustry Association (to S Watanabe), the Research Foundation of the Association for the Progress of New Chemistry (to S Watanabe), the New Energy and Industrial Technology Development Organization (to S Watanabe), and CREST, Japan Science and Technology Agency (to K Makino) We thank Y Takada and T Hirose, Hokkaido.. .Novel L-rhamnose metabolic pathway S Watanabe and K Makino a-ketoglutarate via aKGSA by dl-2-keto-3-deoxypentonate dehydratase and aKGSA dehydrogenase d-KDA dehydratase is involved in this type of archeal d-arabinose metabolism (E73 [14,24]), and the l-arabinose and d-xylose pathways of bacteria and ⁄ or Archaea also contain the homolog (E69, E78, and E83) It was found previously that, in spite of. .. and dehydratases with dl-2-keto-3-deoxyacidsugar but also l-DKDR hydrolase can be assigned as equivalent within the analogous schematic conversions to the ED pathway (Fig 1) In this article, we identified a novel metabolic fate of dl-2-keto-3-deoxyacid -sugar intermediate to pyruvate and hydroxyl-acid through a continuous reaction of dehydrogenase and hydrolase, which is referred to as ‘type III of nonphosphorylated. .. Identification of the missing links in prokaryotic pentose oxidation pathways: evidence for enzyme recruitment J Biol Chem 281, 2737 8–2 7388 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, 261 2–2 623 1566 16 Watanabe S, Kodaki T & Makino K (2006) A novel a-ketoglutaric... as ‘type III of nonphosphorylated sugar pathways’ (Fig 1) The significant insights about metabolic genes illustrated in Fig 1 (modified from a previous study [12]) will be helpful in identifying unknown sugar pathways analogous to the ED and npED pathways and EAT09363) were used to design genomic PCR primers for the amplification of a DNA fragment of SpLRA 1–3 , SpLRA5, and SpLRA6, respectively (Table S2)... Oxidation of D-fucose to D-fucono-d-lactone by a D-aldohexose dehydrogenase J Biol Chem 247, 222 2–2 227 Dahms AS & Anderson RL (1972) D-Fucose metabolism in a pseudomonad II Oxidation of D-fucose to D-fucono-d-lactone by an L-arabino-aldose dehydrogenase and hydrolysis of the lactone by a lactonase J Biol Chem 247, 222 8–2 232 Dahms AS & Anderson RL (1972) D-Fucose metabolism in a pseudomonad III Conversion of. .. 3352 1–3 3536 18 Stephens C, Christen B, Fuchs T, Sundaram V, Watanabe K & Jenal U (2007) Genetic analysis of a novel pathway for D-xylose metabolism in Caulobacter crescentus J Bacteriol 189, 218 1–2 185 19 Watanabe S, Yamada M, Ohtsu I & Makino K (2007) a-Ketoglutaric semialdehyde dehydrogenase isozymes involved in metabolic pathways of D-glucarate, D-galactarate and hydroxy-L-proline: molecular and metabolic . B E B B B Domain B B B B B A B A B E B B A B B B B E b – – 31 34 38 43 47 57 62 67 76 81 – 13 17 b 2 10 21 – c 32 35 39 44 48 50 53 58 63 68 72 77 82 25 14 18 22 c 3 6 28 f 36 41 45 49 51 54 59 64 69 73 78 83 26 15 19 24 e 4 7 29 – 40 – – – – – – – – – – – – – – 23 d – – – L-Glyceraldehyde a – – 30 33 37 42 46 56 61 66 75 80 – 12 16 a 1 8 9 11 27 III III I I PPP PPP PPP ED Fig Novel modified version of nonphosphorylated sugar metabolism – an alternative L-rhamnose pathway of Sphingomonas sp. Seiya Watanabe 1,2,3 and Keisuke