Eur J Biochem 269, 347±357 (2002) Ó FEBS 2002 A b-lysine adenylating enzyme and a b-lysine binding protein involved in poly b-lysine chain assembly in nourseothricin synthesis in Streptomyces noursei Nicolas Grammel1,*, Kvitka Pankevych2, Julia Demydchuk2, Klaus Lambrecht2, Hans-Peter Saluz2, È Ullrich Keller1 and Hans Krugel2 Max-Volmer-Institut fuÈr Biophysikalische Chemie und Biochemie, Fachgebiet Biochemie und Molekulare Biologie, Technische UniversitaÈt Berlin, Germany; 2Department of Cell and Molecular Biology, Hans KnoÈll Institute for Natural Product Research, Jena, Germany Nourseothricins (syn Streptothricins), a group of nucleoside peptides produced by several streptomycete strains, contain a poly b-lysine chain of variable length attached in amide linkage to the amino sugar moiety gulosamine of the nucleoside portion We show that the nourseothricin-producing Streptomyces noursei contains an enzyme (NpsA) of an apparent Mr 56 000 that speci®cally activates b-lysine by adenylation but does not bind to it as a thioester Cloning and sequencing of npsA from S noursei including its ¯anking DNA regions revealed that it is closely linked to the nourseothricin resistance gene nat1 and some other genes on the chromosome possibly involved in nourseothricin biosynthesis The deduced amino-acid sequence revealed that NpsA is a stand-alone adenylation domain with similarity to the adenylation domains of nonribosomal peptide synthetases (NRPS) Further analysis revealed that S noursei contains a b-lysine binding enzyme (NpsB) of about Mr 64 100 which can be loaded by NpsA with b-lysine as a thioester Analysis of the deduced amino-acid sequence from the gene (npsB) of NpsB showed that it consists of two domains The N-terminal domain of  100 amino-acid residues has high similarity to PCP domains of NRPSs whereas the 450-amino-acid C-terminal domain has a high similarity to epimerization (E)-domains of NRPSs Remarkably, in this E-domain the conserved H-H-motif is changed to H-Q, which suggests that either the domain is nonfunctional or has a specialized function The presence of one single adenylating b-lysine activating enzyme in nourseothricin-producing streptomycete and a separate binding protein suggests an iteratively operating NRPS-module catalyses synthesis of the poly b-lysine chain The nourseothricins belong to the family of the streptothricin antibiotics that are produced by various streptomycete strains such as Streptomyces noursei [1] These compounds are nucleoside peptides containing a carbamido-D-gulosamine core, to which a poly b-lysine chain and the unusual amino acid streptolydine are attached in amide and N-glycosidic linkages, respectively (Fig 1) The various members of the group differ in the length of their poly b-lysine chains Streptothricins are potent inhibitors of prokaryotic protein biosynthesis, but are not used therapeutically due to their nephrotoxicity [2] Nourseothricin is currently being used under the name CloNat, and is an effective selective agent for molecular cloning technologies in fungi and plants [3±6] On the other hand, streptothricins are also being tested as fungistatics in agriculture for the treatment of blast disease and other plant diseases [7] Knowledge of the biosynthesis of streptothricins mainly stems from in vivo precursor studies (Fig 1; reviewed in [8]) Thus, streptolydine is derived from arginine [9], gulosamine from glucosamine [10], and b-lysine from a-lysine [11] As the nourseothricins combine the structural features of peptides and nucleosides, their biosynthesis involves quite diverse enzyme activities for sugar biosynthesis, peptide bond formation, glycosylation and the formation of the nourseothricin precursors such as b-lysine and streptolydine The poly (b-lysine) chains of nourseothricins are unique structures as they are made up from identical (b-lysine) amino-acid residues connected to each other with e-(b-lysyl)-peptide bonds and with the chain attached to the amino group of the gulosamine moiety Peptide bond formation in natural products is often catalysed by nonribosomal peptide synthetases (NRPSs), a family of highly conserved enzymes, which are composed of modules each responsible for the activation and incorporation of always one individual Correspondence to H Krugel, Department of Cell and Molecular È Biology, Hans Knoll Institute for Natural Product Research, È Beutenbergstraûe 11, D-07745 Jena, Germany, Fax: + 49 3641 656694, Tel.: + 49 3641 656684, E-mail: hkruegel@pmail.hki-jena.de, or U Keller, Max-VolmerInstitut fur Biophysikalische Chemie und È Biochemie, Fachgebiet Biochemie und Molekulare Biologie, Technische Universitat Berlin, Franklinstrasse 29, D-10587 Berlin, Germany È Tel.: + 49 30 314 25653, E-mail: Ullrich.Keller@TU-Berlin.de Abbreviations: NRPS, nonribosomal peptide synthetase; A-domain, adenylation domain; PCP-domain, peptidyl carrier domain; C-domain, condensation domain; 4¢-Ppan, 4¢-phosphopanthetheine; E-domain, epimerization domain; M-domain, methylation domain *Present address: ActinoDrug Pharmaceuticals GmbH, Hennigsdorf, Germany (Received June 2001, revised 11 October 2001, accepted November 2001) Keywords: nonribosomal peptide synthetase; PCP-domain, b-lysine, nourseothricin, Streptomyces 348 N Grammel et al (Eur J Biochem 269) Ó FEBS 2002 Fig Structure of nourseothricin and its biosynthetic precursors amino acid into a given peptide product [12,13] The sequential order and number of the various modules of a NRPS system determines the sequence and the length of the peptide product The modules consist of domains including the adenylation domain (A-domain) responsible for amino-acid recognition and their activation as an aminoacyl adenylate, and the peptidyl carrier domain (PCP-domain or T-domain) C-terminal to the A-domain providing a covalently bound 4¢-phosphopanthetheine (4¢-Ppan) cofactor for thioester binding of amino-acid substrates and of peptidyl intermediates [14] The third essential domain of a module is the condensation domain (C-domain) located aminoterminally to the adenylation domain which catalyses condensation of amino acid thioester attached to adjacent modules Besides the minimal set of A, T and C domains, modules of NRPS may also harbour epimerization (E) domains, methylation (M) domains, cyclization (Cy) domains instead of the C domain, oxygenation (Ox) domains or reduction (Red) domains These catalyse modi®cation reactions on amino acids or peptidyl intermediates [15] The fact that the poly b-lysine chains of the different nourseothricins consist of identical residues raises the question as to whether the residues are incorporated by a modular peptide synthetase containing several distinct b-lysine modules or whether there is only one module condensing the various b-lysine residues iteratively Another question is how the b-lysines are added to the gulosamine moiety By analogy to some poly amino acids such as folylpoly c-glutamate, an amino-acid polymer produced by bacteria and eukaryotes [16], poly b-lysine synthesis could possibly occur by a mechanism involving activation as b-lysyl phosphate and subsequent ligation of b-lysine residues in an iterative fashion Thus, the condensing enzyme would belong to the ADP-forming amide bond ligase superfamily containing enzymes such as folyl-poly c-glutamate synthetase, UDP-N-acetyl-muramoyl-L-alanine-glutamate ligase, glutathione synthetase or D-Ala-DAla ligase All of these condense carboxylate-containing compounds with a free amino group without covalent binding of the substrate to the enzyme This differs from the nonribosomal thiol template mechanism [17] On the other hand, gene disruption experiments in streptothricin-producing S rochei revealed a gene locus involved in streptothricin biosynthesis with ®ve genes including one encoding resistance against the antibiotic [18] One of the ORFs encodes a protein with similarity to the adenylation domains of peptide synthetases This points to nonribosomal mechanisms of streptothricin biosynthesis However, from the data it was not clear which substrate the enzyme would activate: b-lysine or streptolydine, the latter containing an internal peptide bond To clarify the mechanism of poly b-lysine synthesis during nourseothricin synthesis we set out to isolate the hypothetical poly b-lysine synthetase or its NRPS equivalent from S noursei and to clone the gene We found that S noursei contains a stand-alone A-domain that activates b-lysine by adenylation It was also found that S noursei harbours a protein that after activation speci®cally binds b-lysine as a thioester This protein contains a PCP-domain and a second domain with strong similarity to E-domains of peptide synthetases, which indicates that the poly b-lysine chain of nourseothricins is synthesized by a thiol template mechanism MATERIALS AND METHODS Strains and their cultivation S noursei JA3890b was from the strain collection of the Hans Knoell Institute [19] The strain was maintained on agar slants and cultivated in submerged cultures in medium M79 [20] Mycelia for enzyme preparations were harvested from cultures propagated in 500 mL conical ¯asks containing 100 mL medium at 28 °C for 2±3 days The cultures were cooled on ice, the mycelia were collected by centrifugation at °C, washed in 0.9% NaCl solution and stored at ) 80 °C until use S lividans 1326, from the John Innes Collection, was maintained and cultivated as described previously [21] Ó FEBS 2002 Poly b-lysine assembly in S noursei (Eur J Biochem 269) 349 Chemicals and radiochemicals b-Lysine was kindly provided by U Graefe, Hans Knoll Institute and by P A Frey, University of Wisconsin, Madison, WI, USA [3H]b-Lysine (220 Ciámmol)1) was obtained from Hartmann Analytik, GMbH, Braunschweig, Germany [32P]Tetrasodium pyrophosphate (17.8 Ciámol)1) was from New England Nuclear (NEN) Streptolydinegulosamine was obatined by partial hydrolysis of nourseothricin according to the previously described method [22] The compound was characterized as described previously [23] All other chemicals were of the highest purity commercially available Puri®cation of b-lysine activating enzyme All operations were carried out at °C in a cold room S noursei mycelia (50 g) were suspended in 200 mL buffer B and passed through a French pressure cell at 68 947 kPa The resultant homogenate was treated with  50 lgámL)1 DNAse I (grade II, Sigma) in the presence of 20 mM MgCl2 for h After centrifugation at 30 600 g for 30 min, the supernatant was applied onto a Q-Sepharose FF column (column dimensions 10 ´ cm) previously equilibrated with buffer B (see below) After washing the column with 50 mL of buffer B, the enzyme was eluted with a 200-mL linear gradient from to 0.2 M NaCl in buffer B (5 mL fractions) Fractions with b-lysine-dependent ATP-pyrophosphate exchange activity were pooled and saturated ammonium sulphate was added up to a ®nal saturation of 66% The solution was then left on ice for at least h The resulting suspension was centrifuged as above, the pellet was dissolved in buffer B and applied to a HiLoad 26/60 Superdex 75 pg column (Pharmacia), which had been previously equilibrated with buffer B The ¯ow rate was mLámin)1 and the fraction size was mL Fractions containing b-lysine activating activity were pooled Fig Identi®cation of NpsA by gel ®ltration of b-lysine activating enzyme on Superose 12 Concentrated enzyme (300 lL) from the phenlysuperose step of Table were applied onto a Superose 12 column (Pharmacia) 300 lL fractions were collected The fractionation range from fraction 35±55 is shown (3/4) Absorbance at 280 nm; (bars) activity pattern of the b-lysine-dependent ATP/PPi exchange The inset shows SDS/PAGE (10% polyacrylamide, according to [22]) of 30 lL portions of the indicated fractions Staining was with Coomassie blue The band representing the b-lysine activating enzyme NpsA is denoted by an arrow The pooled enzyme was applied onto an anion exchange column (Mono Q HR5/5, Pharmacia) equilibrated in buffer B and was eluted with a linear gradient from to 0.2 M NaCl in buffer B (¯ow rate mLámin)1, gradient 60 min) Fractions containing enzyme activity were pooled and saturated ammonium sulphate solution was added to a ®nal concentration of 10% The mixture was applied onto a phenyl Superose HR5/5 (Pharmacia) column equilibrated with buffer B containing ammonium sulfate at 10% saturation The column was eluted with a descending gradient of 10 to 0% ammonium sulphate (¯ow rate 0.5 mLámin)1, 45 mL total volume) The b-lysine-activating enzyme eluted at an ammonium sulphate concentration corresponding to 8±9% saturation Fractions containing enzyme were pooled and concentrated in a microconcentrator (Centricon 30, Amicon) Concentrated enzyme was subjected to gel ®ltration chromatography (Superose 12 HR 10/30 column, Pharmacia) using a Smart chromatography system (Pharmacia) The ¯ow rate was 300 lLámin)1 and 200 lL fractions were collected (Fig 2) Active fractions were subjected to SDS/ PAGE After staining, the protein band corresponding to the enzyme was isolated for further analysis Puri®cation of the b-lysine binding protein All operations were carried out in a cold room S noursei mycelia (40 g) suspended in 200 mL of buffer B was passed through a French pressure cell at 68 947 kPa DNAse I (grade II, Sigma) was added at 50 lgáL)1 and MgCl2 at 20 mM and left on ice with stirring for h After centri4 fugation for 20 at 30 600 g, the supernatant was adjusted to a conductivity of mS with water (containing 10 mM dithioerythritol) and applied onto a Q-Sepharose FF column (column dimension 10 cm ´ cm) pre-equilibrated with buffer B After washing the column with 100 mL of buffer B a linear gradient (total volume 200 mL) of 0±0.2 M 350 N Grammel et al (Eur J Biochem 269) NaCl was passed through the column and 5-mL fractions were collected Fractions were assayed by determination of covalent binding of [3H]b-lysine to protein in the presence of the b-lysine activating enzyme, ATP and MgCl2 (see below) Fractions containing b-lysine binding activity were pooled and saturated ammonium sulphate solution was added until 70% saturation After leaving on ice overnight, the suspension was centrifuged as described above Pelleted protein was dissolved in a small volume of buffer B The sample was subjected to gel ®ltration using HiLoad 26/60 Superdex 75 pg column equilibrated with buffer B, and 1-mL fractions were collected Fractions containing the b-lysine binding protein were combined and subjected to anion exchange chromatography on Resource Q (6 mL column, Pharmacia) equilibrated with buffer B A 120-mL gradient (¯ow rate mLámin)1) was applied and mL fractions were collected The active fractions were pooled and brought to 20% ammonium sulfate saturation The solution was applied onto a phenyl Superose HR5/5 (Pharmacia) column equilibrated with buffer B containing ammonium sulphate at 20% saturation and the protein was eluted with a 60-mL gradient (¯ow rate mLámin)1) ranging from 20 to 0% ammonium sulphate saturation The enzyme was eluted at 6% saturation Active fractions were pooled and concentrated to a ®nal volume of 200 lL using a Centricon 30 microconcentrator The sample was then subjected to gel ®ltration on a Superose 12 HR 10/30 column (Pharmacia) previously equilibrated in buffer C at a ¯ow rate of 200 lLámin)1 Fractions (300 lL) were collected and the b-lysine binding protein containing fractions were pooled Active fractions were subjected to analysis by gel electrophoresis Enzyme assays The ATP-pyrophosphate exchange reaction mixture contained mM b-lysine, 2.5 mM ATP, mM MgCl2, 0.1 mM tetrasodium pyrophosphate and ´ 105 c.p.m [32P]tetrasodium pyrophosphate and 10±50 lL of b-lysine activating enzyme fraction in a total volume of 220 lL The mixture was incubated for 10 at 28 °C and stopped by the addition of 0.5 mL charcoal suspension [24] After 10 on ice, the charcoal was collected by suction ®ltration on glass ®ber ®lters, washed once with 35 mL of water and after drying at 80 °C (1 h), the ®lters were counted in a liquid scintillation counter Speci®c activity is de®ned as nkatal, the amount of enzyme catalysing the incorporation of nmol pyrophosphate into ATP per second in the presence of b-lysine The b-lysine binding protein was assayed in a coupled assay with the b-lysine activating enzyme The assay contained 1±2 pkatal of b-lysine activating enzyme, 0.1 mM b-lysine, 0.1 lCi [3H]b-lysine, 18 mM ATP, 33 mM MgCl2 and 5±25 lL of b-lysine binding protein fraction in a total volume of 60 lL After 30 of incubation at 28 °C, mL 7% trichloroacetic acid was added The mixture was left on ice for 30 The precipitated protein was collected on membrane ®lters (ME 30, Schleicher & Schuell), washed with 35 mL of water and after drying, radioactivity was counted in a liquid scintillation counter [25] Ó FEBS 2002 Buffers and solvent systems Buffer B contained 0.1 M Tris/HCl, pH 8.0, mM dithioerythritol, mM benzamidine, mM phenylmethylsulfonyl ¯uoride, mM EDTA Buffer C was the same as buffer B except that it contained 0.05 M Tris/HCl, pH 7.5 Solvent systems for thin layer chromatography of b-lysine were n-butanol/acetic acid/water (4 : : 1,v/v/v;solventsystem I) or isopropanol/acetic acid/water (7 : : 2, v/v/v; solvent system II) Methods of analysis Protein concentrations were determined according to Bradford [26] SDS/PAGE was carried out according to Laemmli [27] Staining of gels was according to standard procedures Radioactivity determinations were by scintillation counting with a scintillation cocktail (Quicksafe A, Zinsser Analytic) [25] Thin-layer chromatograms (Silica gel 60, Merck, Darmstadt) were autoradiographed by exposure to Kodak X-ray ®lm (Biomax MS) b-Lysyl thioester was analysed by performic acid treatment of trichloroacetic acid precipitated enzyme thioester as described previously [25] Protein sequence determinations Peptide sequences were determined with a Procise peptide Sequencer (Applied Biosystems) Bands from SDS/PAGE separations of the b-lysine activating enzyme blotted onto poly(vinylidene di¯uoride) membrane were visualized with Ponceau S, cut out and directly sequenced In the case of the b-lysine binding protein, bands in gels were visualized by Coomassie staining Gel pieces were cut out and subjected to in-gel digestion with trypsin as described previously [28] After elution, the tryptic peptide mixture was separated by HPLC (lRPC C2/C18 column, Pharmacia) with acetonitrile/water gradients in the presence of tri¯uoroacetic acid Well resolved peaks were subjected to sequencing In the case of the b-lysine binding protein six peptide sequences were obtained which were used for the design of various oligonucleotide primers for PCR The pair pcp 15, GAG CACGGCMGRGAGGAGGC/PCP; 6, SGCSARGTG SCCSACSGT gave a clone encoding a partial sequence of NpsB DNA manipulations All DNA manipulations were performed according to published procedures [29] In particular, genomic DNA of S noursei was prepared from lysozyme-digested mycelium by phenol/chloroform puri®cation as described previously [21] To construct a genomic library, the DNA was partially digested with Sau3A DNA fragments in the size range from 10 to 20 kb were ligated to BamHI-cleaved lambda phage vector arms (Lambda GEM-11 Packagene system, Promega) Screening for recombinant phages carrying nourseothricin biosynthesis genes was initially carried out by hybridization of plaques with the nourseothricin resistance gene nat1 [4±6], and in the later course of this work, a fragment of the b-lysine-binding enzyme gene was used The latter fragment had been generated by PCR from chromosomal DNA of S noursei using primers derived from two internal peptide sequences of the b-lysine binding protein Ó FEBS 2002 Poly b-lysine assembly in S noursei (Eur J Biochem 269) 351 The labelling of the probes was with the digoxiginenlabelling kit from Boehringer, Mannheim Hybridizing plaques were picked, puri®ed and analysed by restriction mapping and hybridization along with chromosomal DNA as control From each screening, one representative phage (phage ph1-2 and phage ph41, respectively) was chosen for subclone preparation using pUC118 or pBlueScriptKS Sequencing was performed on a LiCor automated system Plasmids The plasmid for expression of npsA was pDW5 (K Weber, J Demydchuk, U Peschke, unpublished results) Plasmids for subcloning and sequencing were pUC118 and pBlueScriptKS Nucleotide sequence accession number The DNA sequence data have been deposited in the EMBL nucleotide sequence database under accession nos AJ315729 (npsA) and AJ315730 (npsB) RESULTS b-Lysine activation in S noursei Peptide synthetases activate their amino-acid substrates by adenylation with subsequent covalent binding to a PCP via a thioester linkage To seek b-lysine adenylating activity possibly involved in nourseothricin biosynthesis, protein extracts of S noursei actively synthesizing nourseothricin were fractionated by anion exchange chromatography on Q-Sepharose FF matrix (Pharmacia) Assay of fractions from such separations for b-lysine-dependent ATP-pyrophosphate exchange revealed a single peak of enzyme activity (not shown) The enzyme was further puri®ed by gel ®ltration on Superdex 75 pg, anion exchange chromatography on Mono Q HR5/5 and hydrophobic chromatography on phenyl Superose In all these separations the activity was found in one single peak The ®nal puri®cation was 50-fold (Table 1) SDS/PAGE analysis of enzyme from the last puri®cation step revealed several other protein bands (not shown) As further attempts to purify the enzyme activity to homogeneity failed, we subjected the enzyme from the phenyl Superose step to gel ®ltration on Superose 12 HR The activity of each fraction was determined and each fraction was subjected to SDS/PAGE analysis By this procedure, activity could be correlated with the intensity of a particular band of 56 kDa (Fig 2) The enzyme was named NpsA (nourseothricin peptide synthetase A) Microsequencing of this band yielded the N-terminal sequence: MESS ASSFLEPFFDVXR Characterization of the b-lysine activating enzyme (NpsA) from S noursei Passing NpsA through a calibrated Superdex 75 pg gel ®ltration column revealed that the enzyme has an Mr between 58 000 and 60 000 This ®tted with the estimated molecular mass of NpsA in its denatured form (Fig 2) and also indicates that the native form of the enzyme is a monomer The puri®ed enzyme when incubated with tritium-labelled b-lysine, ATP and MgCl2 did not bind the labelled amino acid covalently, which indicates that the enzyme most probably represented a stand-alone A-domain without a PCP-domain The enzyme's substrate speci®city was determined by measuring the ATP/pyrophosphate exchange in the presence of different amino acids structurally related to b-lysine The enzyme did not activate a-lysine or arginine (b-arginine not tested) This indicates that the active site of the enzyme can strictly distinguish between an a-amino and a b-amino group of lysine Other b-amino acids such as b-alanine or b-aminobutyric acid, c-aminobutyric acid and e-amino caproic acid were not activated Thus, the activating enzyme appears to be strictly speci®c for b-lysine The strict speci®city of the enzyme for b-lysine strongly suggests that it is part of the nourseothricin synthesizing enzyme system No other b-lysine activating enzymes were detected in S noursei Cloning of the NpsA gene Phages ph41 and phN6 are overlapping clones obtained from screening of a phage library of S noursei DNA using the nourseothricin resistance gene nat1 as a probe (Fig 3) nat1 has been previously cloned from S noursei by its property to confer resistance to nourseothricin in foreign streptomycetes [4] The gene encodes a nourseothricinacetylase (NatI) which speci®cally monoacetylates the b-lysine chain of nourseothricin which makes this compound antibiotically inactive It is known that most, if not all, antibiotic biosynthesis gene clusters contain resistance genes against their own antibiotic [31] We therefore concluded that the gene for the b-lysine activating enzyme Table Puri®cation of the b-lysine activating enzyme NpsA from Streptomyces noursei Cells (50g) from a 72-h culture of S noursei was used Puri®cation was based on ATP-pyrophosphate exchange dependent on the presence of b-lysine One nkatalámol)1 is the amount of enzyme catalysing the exchange of nmol of pyrophosphate into ATP per second ND, not determined Puri®cation step Volume (mL) Protein (mg) Activity (nkatal) Speci®c activity (pkatalámg)1) Yield (%) Puri®cation (fold) Crude extract Q-Sepharose FF (NH4)2SO4 60% Superdex 75 HR MonoQ Phenylsuperose 120.0 67.0 2.5 8.0 4.0 2.0 1800 101 88 28 1.3 ND 4.197 4.047 1.890 1.875 0.750 ND 41.6 46.0 67.5 468.8 577.0 ND 100 96 45 45 18 ND 1.0 1.1 1.6 11.3 50.0 352 N Grammel et al (Eur J Biochem 269) Ó FEBS 2002 11 Fig Map of the sequenced region containing the nourseothricin resistance gene nat1 and the gene npsA encoding the b-lysine activating enzyme NpsA (A) The region represents the overlapping region of phages phP41 and phN6 Arrowheads indicate the orientation and relative length of the sequenced orfs Based on the similarities of their deduced amino-acid sequences with proteins in the database, ORFs A±E are proposed to encode proteins with the following functions: ORF A (acylase), ORF B (thioesterase), ORF C (phosphotransferase), ORF D (unknown), ORF E (regulator) The same orfs in a similar arrangement have been shown to be present in a region of the streptothricin biosynthesis gene cluster of S rochei [27] (B) The strategy of expression cloning of npsA is shown in the lower part B of the ®gure The EcoRI fragment was subcloned in pBluescript and cloned as a XbaI±HindIII fragment into plasmid pDW5 as described in the text Vegp40 denotes the position of the veg promoter of B subtilis [25] could possibly lie in the same region of the S noursei chromosome as the resistance gene Chromosome walking (by subcloning various BamHI or NotI fragments into pBlueScript and subsequent sequencing) on the overlapping region of the DNA inserts of phP41 and phN6 revealed several ORFs in the 3¢ region of nat1 (Fig 3) One ORF of 1518 bp was interesting because it encoded a hypothetical protein of 506 residues with a deduced Mr of 53 kDa, which is in the range of the estimated molecular mass of NpsA The deduced N-terminal sequence of this enzyme is identical to the sequence determined by microsequencing of NpsA (see above), which supports that this gene (designated npsA) is the gene encoding the b-lysine activating enzyme NpsA has a typical Streptomyces codon usage with a strong bias for high G/C content (> 90%) in the third codon positions of the gene The overall G/C content of the gene is 75% Analysis of the deduced amino-acid sequence of NpsA revealed high similarity with the adenylation domains of various NRPSs NpsA possesses all the 10 conserved signature sequences A1 to A10 characteristic of the adenylation domains of NRPS [15] Remarkably, the enzyme has 95% amino-acid sequence identity with SttA from the biosynthesis gene cluster of streptothricin in S rochei, which con®rms the previously proposed role of the enzyme as a b-lysine activating enzyme [18] An alignment of the amino-acid residues of the adenylate binding pocket of NspA as well as of SttA with consensus sequences derived from the relevant binding residues in the amino-acid binding pocket of the phenylalanine A-domain of gramicidin S synthetase [32,33] showed their similarity to the binding pockets of A-domains, which are known to activate positively charged amino acids such as ornithine and lysine In particular the characteristic Asp in position 239 of NpsA indicated a strong relationship to the lysine-activating Table Speci®city determining residues in the binding pocket of NpsA Alignment of the speci®city determining residues of NpsA adenylation domain with several adenylation domains activating amino acids with positively charged side chains NpsA (b-lysine, this work), SttA (adenylating enzyme from S rochei [27]), BacB_M1 (a-lysine, module of bacitracin synthetase B, accession no AAC06347), BacB_M2 (a-ornithine, module of bacitracin synthetase B, Acc.No AAC06347), GrsB_M3 (a-ornithine, module of gramicidin synthetase B, accession no CAA43838) The speci®city conferring residues were aligned according to the method of Stachelhaus et al [28] Position in amino-acid binding pocket Enzyme Amino acid 234 235 236 239 278 299 301 322 330 331 NpsA SttA BacB_M1 BacB_M2 GrsB_M3 b-Lys b-Lys a-Lys a-Orn a-Orn G G F F F D D D D D T T A V V E E E G G G G S E E V V I I I G G G G G T T S S S L L V V L V V C D I Ó FEBS 2002 Poly b-lysine assembly in S noursei (Eur J Biochem 269) 353 module of bacitracin synthetase (Table 2) Interestingly, the universally conserved Phe234 of NRPS A-domains is changed into a glycine residue in both NspA and SttA, which may result in a change of conformation of the Asp235 side chain possibly binding the unusual b-amino-acid substrate Expression of npsA in S lividans To con®rm the identity of the gene npsA as the gene encoding NpsA, a 3-kb EcoRI fragment from the phage clone ph41 (Fig 3) encompassing the entire npsA gene was ligated to EcoRI-cleaved Bluescript vector and after excision as a HindIII±XbaI fragment was ligated into HindIII±XbaIcleaved pDW5, a derivative of pWHM4, under the control of the veg promoter from Bacillus subtilis [34] (Fig 3) Transformation of S lividans by the plasmid containing the cloned gene resulted in strain S lividans W5 Crude extracts of strain W5 were prepared as for S noursei and fractionated on Q-Sepharose FF Testing the fractions clearly revealed the presence of the b-lysine activating activity of NpsA in this S lividans strain, which was missing in a control strain containing plasmid pDW5 (not shown) These data unambiguously indicate that the npsA gene encodes the b-lysine activating enzyme NpsA Detection and puri®cation of a b-lysine binding protein in S noursei A prerequisite in peptide bond formation between amino acids in nonribosomal systems is the covalent activation of amino-acid residues as enzyme-linked thioesters [12,13] As the b-lysine activating enzyme is a stand-alone adenylation domain lacking a PCP-domain, we set out to identify the missing amino acyl or peptidyl carrier protein which would represent the rest of the missing part of the putative b-lysine module Using radiolabelled b-lysine as substrate, we tested fractions of protein extracts from S noursei for the presence Fig Covalent labelling of NpsB with radioactive b-lysine Partial puri®ed NpsB (phenylSuperose step, see Materials and methods) was incubated with NpsA, radioactive b-lysine and ATP The reaction mixtures were incubated at 28 °C for 30 mL 5% trichloroacetic acid was added and precipitated protein was recovered by centrifugation Protein was subjected to SDS/PAGE (10% SDS/polycarylamide slab) The gel was subjected to autoradio¯uorography using Amplify solution (Amersham) according to the manufacturer's instructions Auto¯uorography was for weeks The complete assay mixture contained 0.1 mM b-lysine, 0.1 lCi [3H]b-lysine, 18 mM ATP, 33 mM MgCl2, pkatal NpsA and lL phenylsuperose fraction of NpsB (lane A); lane B with omission of NpsA, lane C with omission of ATP, lane D with omission of NpsB Left panel: Coomassie Blue-stained gel Right panel: Auto¯uorograph of same gel of a protein that would bind b-lysine covalently after its activation as adenylate by NpsA Separation of a crude extract of S noursei on a Q Sepharose FF and testing fractions for covalent binding of radioactive b-lysine in the presence of NpsA and ATP revealed a peak of b-lysinebinding activity Gel ®ltration on a Superdex 75 pg column revealed that the binding protein has a surprisingly high Mr ( 70 000 Da) which in view of the small sizes of PCPdomains ( 100 residues) of NRPS indicates that this protein must be a multimer or must harbour additional functions besides binding b-lysine as a thioester The b-lysine binding protein eluted independently of the b-lysine activating enzyme from the Q Sepharose FF column which indicates that these two enzymes not form a stable complex with each other (not shown) To test the nature of the covalent bond between the binding protein and b-lysine, the b-lysine binding protein was charged with radioactive b-lysine and the covalent enzyme±substrate complex was subjected to performic acid oxidation The radioactive b-lysine released from the enzyme was identi®ed by thin layer chromatography (using solvent systems I and II) Treatment of charged protein with formic acid released no b-lysine indicating that b-lysine is indeed bound to the protein as a thioester Analysis by SDS/PAGE at each stage of puri®cation (see Materials and Methods) of the b-lysine binding protein revealed enrichment of a prominent band of  70 kDa (Fig 4A) To identify this band as the b-lysine binding protein, the native enzyme was loaded with radioactive b-lysine in the presence of NpsA and ATP and subjected to SDS/PAGE Figure shows that the 70-kDa band was speci®cally labeled with b-lysine This reaction was ATP-dependent and also dependent on the presence of the b-lysine activating enzyme NpsA The protein was named NpsB Attempts to demonstrate the formation of b-lysyl±b-lysine or poly (b-lysine) in incubations containing puri®ed NpsA and NpsB with ATP and [3H]b-lysine failed No evidence for the formation of such products was obtained either as free or enzyme bound material Moreover, 354 N Grammel et al (Eur J Biochem 269) incubation of NpsA and NpsB with ATP, [3H]b-lysine and streptolydine-gulosamine did not lead to detectable synthesis of a new compound dependent on streptolydine/gulosamine By contrast, the thin layer chromatograms of reaction mixtures showed only formation of a free compound during these incubation with an Rf value much higher than that of b-lysine The formation of this compound was ATP-dependent and strictly dependent on the presence of either NpsA or NpsB The possibility that this compound must be the spontaneous cyclization product of the b-lysine thioester (cyclo-b-lysine) could not be determined due to the lack of authentic reference material Cloning of the gene of the b-lysine binding protein Total protein from the last puri®cation step of NpsB was subjected to preparative SDS/PAGE and the band representing the b-lysine binding protein was in-gel digested with trypsin After HPLC separation, six tryptic peptides were sequenced Each of the resultant sequences was used to design PCR primers for both strands The primers were used in PCR in all possible combinations to amplify the gene using chromosomal DNA of S noursei as template One primer pair (see Materials and methods) yielded a PCR product with sequences corresponding to the amino-acid sequence of the binding protein This indicates that the clone represents a partial sequence of the gene of the b-lysine binding protein The PCR fragment was in turn used as a probe in plaque hybridization screening of our S noursei phage library From one hybridizing phage, Ph1-2, a 15-kb insert was obtained (not shown) Narrowing down the hybridizing region by restriction mapping and Southern analysis led to the subcloning of a 6.8-kb BamHI fragment, which was partially sequenced Analysis of the sequence revealed three ORFs as shown in Fig The central gene encoded a protein with 606 amino acids of a calculated Mr=64 100, which contained all of the six internal sequences obtained from microsequencing of the peptide Ó FEBS 2002 fragments, thus con®rming that the gene is npsB Analysis of the deduced amino-acid sequence indicated that the protein is composed of two distinct domains The ®rst domain located between amino-acid residues 40±120 has similarity with various ACP- and PCP-domains of polyketide synthases and peptide synthetases The invariant serine residue representing the 4¢-phosphopanthetheine attachment site is located at amino-acid position 88 The second domain of NpsB located carboxyterminal to the ACP-domain from amino-acid residues 150±550 has similarity with condensation and epimerization domains of a number of peptide synthetases (Fig 5) In particular, the sequence from aminoacid residues 286±296 (HQLAFDMVS) is reminiscent of the signature sequences C3 (HHxISDGxS) or E2 (his-motif) (HHxxxDxVSWxIL) of the C-domains and E-domains of various peptide synthetases, respectively [15] Moreover, in a part of the protein C-terminal to the H-H-motif, all of the ®ve conserved motifs E3 to E7 (in the nomenclature of Konz & Marahiel [15]) of E-domains are present Interestingly, the second His in the C3 or E2 motifs, which was shown to be critical both for the condensation of amino acids in C-domains [35] and the epimerization of amino acids in E-domains [36] in NpsB, is replaced by glutamine suggesting a natural mutation from His to Gln in that sequence As the con®guration of the b-lysine residues in nourseothricin is L and the b-lysine substrate used here was also in the L con®guration (obtained by acid hydrolysis of nourseothricin), the function of this epimerization domain is dif®cult to discern Also, it is not known whether H-Q-versions of the E2/C3 signature sequences are functionally active Sequencing of the upstream region of the npsB revealed an ORF encoding a carbamoyltransferase and further upstream an IS-like sequence The vicinity of a gene encoding a carbamoyltransferase directly relates to the carbamoyl group at the C-5 hydroxyl group of the gulosamine of nourseothricin and is a further hint that the cloned region of the S noursei chromosome with the b-lysine binding protein gene and the carbamoyltransferase Fig Map of a 6.8-kB BamHI fragment from the S noursei chromosome carrying the gene npsB encoding the b-lysine binding protein NpsB The sequenced region spans from the indicated (asterisk) SalI to the BamHI site on the left border of the fragment Arrows indicate the orientation and relative length of the sequenced orfs Sequencing revealed the gene npsB (identi®ed by comparison with sequences of tryptic peptides derived from NpsB), an orf (orf1) with a deduced amino-acid sequence showing homology to carbamoyltransferases, and a IS-like sequence In the lower part of the ®gure is shown schematically the structure of the NpsB protein with the two-domain arrangement consisting of a PCP-domain and a domain with similarity to E-domains of NRPSs Ó FEBS 2002 Poly b-lysine assembly in S noursei (Eur J Biochem 269) 355 gene are part of the nourseothricin biosynthetic gene cluster It is noteworthy, that no overlapping clones connecting the inserts of phage ph41 and Ph1-2 were found In fact, the two inserts appear to be located at a distance of more than 20 kb on the chromosome of S noursei suggesting a separation of the biosynthetic genes in two partial gene clusters DISCUSSION Streptothricins are nucleoside peptides that contain a poly b-lysyl chain which may vary from three to seven residues in length and which is attached to the 2-amino group of the gulosamine moiety of the antibiotic via an amide linkage As yet, a total cell-free synthesis of nourseothricins has not been accomplished, nor have partial enzyme activities from S noursei been characterized The poly(b-lysine) chain is a unique example of a poly amino-acid chain in a secondary metabolite A number of poly(amino acids) such as folylpoly c-glutamate play roles in the housekeeping functions of their producer cells and like the muramyl peptide chains, the tripeptide glutathione or the D-Ala-D-Ala dipeptides of the bacterial cell walls are formed by amino-acid ligases such as UDP-N-acetyl-muramoyl-L-alanine-glutamate ligase, glutathione synthetase or D-Ala-D-Ala ligase [37] They condense carboxylate-containing compounds via the intermediacy of acylphosphates as in the case of formation of the D-AlaD-Ala dipeptide [38] Remarkably, in several compounds synthesized by this mechanism, unusual peptide bonds sych as the c-glutamyl peptide bond also occur as in the muramyl peptides and glutathione As in the poly(b-lysyl) chains, the peptide bond is also unusual (x-b-lysyl) and no poly(aminoacid) synthetases operating via the thiol template mechanism have been described as yet, but it could not be excluded a priori that poly b-lysyl synthesis would occur via a ligase7 like mechanism The data presented here, however, show that the poly(b-lysine) chain of nourseothricin must be synthesized by an NRPS-like system in a mechanism that uses NspA, a stand-alone b-lysine adenylating enzyme (A-domain) and NspB, a b-lysine binding protein consisting of a PCPdomain and a domain with similarity to E-domains of peptide synthetases NspA is unique because of its extraordinarily exclusive substrate speci®city for b-lysine, which contrasts the relaxed speci®city of most NRPS The sequence of NspA is almost identical with that of SttA the enzyme which has been shown previously to be involved in the biosynthesis of streptothricins in S noursei [18] The speci®city-conferring residues of the substrate binding pocket of NspA (and SstA) display a sequence with similarity to that of domains known to activate ornithine, diaminobutyric acid, hydroxyornithine or lysine but with a substantial change of the highly conserved Phe234 present in all NRPS A-domains into a glycine which may lead to alteration of the overall conformation ofthe active site pocket which may lead to the selective binding of b-lysine to this pocket (Table 2) For the condensation of the b-lysine residues, covalent attachment to a PCP-domain in thioester linkage appears to be necessary similarly to other NRPS systems Accordingly, NspA ef®ciently loads NspB with b-lysine in thioester linkage Stand-alone adenylation domains in nonribosomal peptide synthesis have been described for various biosyn- thesis systems, such as of the aryl peptide lactones, the arylsiderophore peptides or in the case of D-alanyl-lipoteichoic acid [39±43], where they activate aromatic carboxylic acids or an amino acid such as alanine as adenylates, which in turn are loaded to speci®c PCP domains These PCPdomains are either alone-standing PCPs, as in the biosynthesis of actinomycin [39] and D-alanyl-lipoteichoic acid, or they are fused to protein domains catalysing another step of the same pathway as in EntB, the PCP for the 2,3dihydroxybenzoic acid in enterobactin synthesis [40] Usually, the PCP-bound carboxylates are condensed with the next amino acids in the biosynthetic sequence by the action of C-domains or Cy-domains forming part of the downstream module Cy-domains not only catalyse condensations between serine, threonine or cysteine residues with upstream thioester-activated amino acids but after condensation also cyclize their substrate to the corresponding oxazoline and thiazoline, respectively [44] In contrast to these examples, the b-lysine-binding PCPdomain described here is fused to a domain with similarity to the E-domains of NRPS, which suggests a different mechanism E-domains in NRPS are always located downstream of PCP-domains and catalyse the conversion of the amino acid or peptidyl intermediate tethered to the PCP-domain from the L to the D con®guration In NRPSs which not epimerize their substrates, C-domains are also always directly located downstream of PCP-domains The mechanistic basis for both E-domains and C-domains is similar They both contain a double His-motif (E2 and C3, respectively) from which the second His of the E-domain is postulated to remove a proton either from the a-carbon of the thioester-activated amino acid or peptidyl intermediate leaving a carbanion intermediate ready for attack by a proton in an Sn2 mechanism [44] In the C3 signature sequence of C-domains, the second His is thought to remove a proton from the amino nitrogen of the acceptor aminoacid in the condensation process [36] Thus, the signi®cance of the E-domain described here is not clear because the second His in the E2 is missing the motif being changed into a H-Q, which would suggest that the E-domain of NspB would be nonfunctional On the other hand, Cy-domains that are present in NRPS catalysing the condensation of a serine, threonine or cysteine residue with an upstream residue under subsequent heterocycle formation have a modi®ed His-motif with only one H which leaves the possibility open for a specialized function of the modi®ed E-domain described here PksF [45] and PpsE [46] encoded by genes from the genomes of B subtilis and M tuberculosis, respectively, are other examples of E-domains in which the second His of the E2-sequence is changed into Q and A, respectively It has not yet been tested whether these E-domains function as E- or C-domains Moreover, the modi®ed E2 motif suggests some signi®cance in the light of the fact that in NpsB an epimerization by the established mechanism [36] of proton abstraction from a-carbon would remain undetectable, because the a-carbon of b-lysine is not asymmetric Thus, if the E-domain of NpsB would have a function in nourseothricin biosynthesis, it could be speculated that this enzyme might catalyse b-lysine condensation in conjunction either with a C-domain or condensing enzyme As yet, we have not been able to demonstrate the formation of a b-lysyl-b-lysine with NpsA and NpsB nor the transfer of b-lysine to gulosamine, which rules out a direct 356 N Grammel et al (Eur J Biochem 269) function of the E-domain of NspB as a condensing domain Interestingly, Stachelhaus et al reported the involvement of the E-domain of GrsA (PheATE) in conjunction with the ProCAT module of GrsB in the condensation of phenylalanine with proline [35] This transfer role was independent from the presence or absence of the second His in the E2-motif of that E-domain Thus, one has to consider the possibility of an additional factor operating in the condensation reaction between the b-lysine residues of the poly(b-lysine) chain as well as the condensation reaction of carboxyl activated b-lysine with the gulosamine moiety The fact, that these condensation reactions involve the participation of only one single stand-alone A-domain and a single binding protein NspB points to an iteration of these two proteins in the assembly line of poly b-lysine chains in nourseothricin biosynthesis This could either take place by the formation of a multimeric complex between the two enzymes or by an iterative mechanism of poly b-lysine chain formation on a single NspA/NspB module To address these questions as well as the true function of the E-domain of NpsB in the condensation of b-lysine residues with each other and also with the gulosamine moiety of nourseothricin, future studies of nourseothricin biosynthesis and the cloning and sequencing of more genes in the cluster 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tuberculosis from the complete genome sequence Nature 393, 537±544  ... that the poly (b-lysine) chain of nourseothricin must be synthesized by an NRPS-like system in a mechanism that uses NspA, a stand-alone b-lysine adenylating enzyme (A- domain) and NspB, a b-lysine. .. b-lysines are added to the gulosamine moiety By analogy to some poly amino acids such as folylpoly c-glutamate, an amino-acid polymer produced by bacteria and eukaryotes [16], poly b-lysine synthesis. .. that S noursei contains a stand-alone A- domain that activates b-lysine by adenylation It was also found that S noursei harbours a protein that after activation speci®cally binds b-lysine as a