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• W2055374672 abstract "(3R,5R)-Clavulanic acid, a clinically used inhibitor of serine β-lactamases, is produced by fermentation of Streptomyces clavuligerus. The early steps in clavulanic acid biosynthesis leading to the bicyclic β-lactam intermediate (3S,5S)-clavaminic acid have been defined. However, the mechanism by which (3S,5S)-clavaminic acid is converted to the penultimate intermediate (3R,5R)-clavaldehyde is unclear. Disruption of orf15 or orf16, of the clavulanic acid biosynthesis gene cluster, blocks clavulanic acid production and leads to the accumulation of N-acetyl-glycyl-clavaminic acid and N-glycyl-clavaminic acid, suggesting that these compounds are intermediates in the pathway. Two alternative start codons have been proposed for orf17 to encode for two possible polypeptides, one of which has 92 N-terminal residues less then the other. The shorter version of orf17 was successfully expressed in Escherichia coli and purified as a monomeric protein. Sequence analyses predicting the ORF17 protein to be a member of the ATP-grasp fold superfamily were supported by soft ionization mass spectrometric analyses that demonstrated binding of ATP to the ORF17 protein. Semisynthetic clavaminic acid, prepared by in vitro reconstitution of the biosynthetic pathway from the synthetically accessible intermediate proclavaminic acid, was shown by mass spectrometric analyses to be converted to N-glycyl-clavaminic acid in the presence of ORF17, ATP, and glycine. Under the same conditions N-acetyl-glycine and clavaminic acid were not converted to N-acetyl-glycyl-clavaminic acid. The specificity of ORF17 as an N-glycyl-clavaminic acid synthetase, together with the reported accumulation of N-glycyl-clavaminic acid in orf15 and orf16 disruption mutants, suggested that N-glycyl-clavaminic acid is an intermediate in clavulanic acid biosynthesis. (3R,5R)-Clavulanic acid, a clinically used inhibitor of serine β-lactamases, is produced by fermentation of Streptomyces clavuligerus. The early steps in clavulanic acid biosynthesis leading to the bicyclic β-lactam intermediate (3S,5S)-clavaminic acid have been defined. However, the mechanism by which (3S,5S)-clavaminic acid is converted to the penultimate intermediate (3R,5R)-clavaldehyde is unclear. Disruption of orf15 or orf16, of the clavulanic acid biosynthesis gene cluster, blocks clavulanic acid production and leads to the accumulation of N-acetyl-glycyl-clavaminic acid and N-glycyl-clavaminic acid, suggesting that these compounds are intermediates in the pathway. Two alternative start codons have been proposed for orf17 to encode for two possible polypeptides, one of which has 92 N-terminal residues less then the other. The shorter version of orf17 was successfully expressed in Escherichia coli and purified as a monomeric protein. Sequence analyses predicting the ORF17 protein to be a member of the ATP-grasp fold superfamily were supported by soft ionization mass spectrometric analyses that demonstrated binding of ATP to the ORF17 protein. Semisynthetic clavaminic acid, prepared by in vitro reconstitution of the biosynthetic pathway from the synthetically accessible intermediate proclavaminic acid, was shown by mass spectrometric analyses to be converted to N-glycyl-clavaminic acid in the presence of ORF17, ATP, and glycine. Under the same conditions N-acetyl-glycine and clavaminic acid were not converted to N-acetyl-glycyl-clavaminic acid. The specificity of ORF17 as an N-glycyl-clavaminic acid synthetase, together with the reported accumulation of N-glycyl-clavaminic acid in orf15 and orf16 disruption mutants, suggested that N-glycyl-clavaminic acid is an intermediate in clavulanic acid biosynthesis. The bicyclic β-lactam clavulanic acid is a potent inhibitor of Class A serine β-lactamases (penicillinases) and is used clinically in combination with semisynthetic penicillins such as amoxicillin (1Brown A.G. Butterworth D. Cole M. Hanscomb G. Hood J.D. Reading C. Rolinson G.N. J. Antibiot. (Tokyo). 1976; 29: 668-669Crossref PubMed Scopus (406) Google Scholar, 2Baggaley K.H. Brown A.G. Schofield C.J. Nat. Prod. Rep. 1997; 14: 309-333Crossref PubMed Scopus (137) Google Scholar). Clavulanic acid is one of a family of clavams produced as secondary metabolites by Streptomyces clavuligerus, but it is unusual among naturally occurring clavams in that it possesses the 3R,5R stereochemistry required for reaction with penicillin-binding proteins and β-lactamases. Although clavulanic acid has only eight carbons and two chiral centers, its lability and density of functionalization renders its preparation via chemical synthesis difficult (2Baggaley K.H. Brown A.G. Schofield C.J. Nat. Prod. Rep. 1997; 14: 309-333Crossref PubMed Scopus (137) Google Scholar, 3Townsend C.A. Curr. Opin. Chem. Biol. 2002; 6: 583-589Crossref PubMed Scopus (48) Google Scholar). Instead, it is commercially isolated from fermentations of S. clavuligerus. There has been interest in clavam biosynthesis with a view to optimizing the fermentation of clavulanic acid as well as for engineering the pathway to produce new derivatives with a broad spectrum of antibacterial and β-lactamase inhibitory activities. The clavulanic acid biosynthesis gene cluster in S. clavuligerus is currently thought to comprise ∼18 genes that are directly involved in its biosynthesis, transport, and regulation (Fig. 1a, or fs 2–19) (4Mellado E. Lorenzana L.M. Rodriguez-Saiz M. Diez B. Liras P. Barredo J.L. Microbiology (Reading). 2002; 148: 1427-1438Crossref PubMed Scopus (56) Google Scholar, 5Li R. Khaleeli N. Townsend C.A. J. Bacteriol. 2000; 182: 4087-4095Crossref PubMed Scopus (50) Google Scholar, 6Hodgson J.E. Fosberry A.P. Rawlinson N.S. Ross H.N. Neal R.J. Arnell J.C. Earl A.J. Lawlor E.J. Gene (Amst.). 1995; 166: 49-55Crossref PubMed Scopus (51) Google Scholar, 7Jensen S.E. Alexander D.C. Paradkar A.S. Aidoo K.A. Baltz R.H. Hegeman G.D. Skatrud P.L. Industrial Microorganisms: Basic and applied Molecular Genetics. American Society for Microbiology, Washington, D. C.1993: 169-176Google Scholar). Another differently regulated clavam gene cluster in S. clavuligerus has also been identified that contains a minimum of four genes homologous to orfs 2, 3, 4, and 6 of the better characterized cluster (8Jensen S.E. Wong A. Griffin A. Barton B. Antimicrob. Agents Chemother. 2004; 48: 514-520Crossref PubMed Scopus (19) Google Scholar, 9Tahlan K. Park H.U. Wong A. Beatty P.H. Jensen S.E. Antimicrob. Agents Chemother. 2004; 48: 930-939Crossref PubMed Scopus (44) Google Scholar). Furthermore, cas1 (clavaminic acid synthase 1), a homologue of cas2 (orf5), is located elsewhere in the genome (10Marsh E.N. Chang M.D. Townsend C.A. Biochemistry. 1992; 31: 12648-12657Crossref PubMed Scopus (79) Google Scholar, 11Paradkar A.S. Jensen S.E. J. Bacteriol. 1995; 177: 1307-1314Crossref PubMed Google Scholar) and is flanked by genes associated with the production of other (3S,5S)-clavam metabolites (12Mosher R.H. Paradkar A.S. Anders C. Barton B. Jensen S.E. Antimicrob. Agents Chemother. 1999; 43: 1215-1224Crossref PubMed Google Scholar). Steps in the pathway leading to (3S,5S)-clavaminic acid, the proposed branch point between the biosynthesis of clavulanic acid and the (3S,5S)-clavams (13Egan L.A. Busby R.W. IwataReuyl D. Townsend C.A. J. Am. Chem. Soc. 1997; 119: 2348-2355Crossref Scopus (40) Google Scholar), have been characterized (Fig. 1b), and the enzymes that catalyze them have been identified. In the first step, l-arginine and d-glyceraldehyde-3-phosphate react to give N2-(2-carboxyethyl)arginine in a thiamin diphosphate-dependent reaction catalyzed by N2-(2-carboxyethyl)arginine synthase (CEAS) 2The abbreviations used are: CEASN2-(2-carboxyethyl)arginine synthaseGCASN-glycyl-clavaminic acid synthetase (ORF17)BLSβ-lactam synthetaseCASclavaminic acid synthasePAHproclavaminate amidino hydrolase2-OG2-oxoglutarateLC-MSliquid chromatography-mass spectrometryESI-MSelectrospray ionization MS.2The abbreviations used are: CEASN2-(2-carboxyethyl)arginine synthaseGCASN-glycyl-clavaminic acid synthetase (ORF17)BLSβ-lactam synthetaseCASclavaminic acid synthasePAHproclavaminate amidino hydrolase2-OG2-oxoglutarateLC-MSliquid chromatography-mass spectrometryESI-MSelectrospray ionization MS. (14Khaleeli N. Li R.F. Townsend C.A. J. Am. Chem. Soc. 1999; 121: 9223-9224Crossref Scopus (81) Google Scholar). β-Lactam formation is catalyzed by β-lactam synthetase (BLS) (15Bachmann B.O. Li R. Townsend C.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9082-9086Crossref PubMed Scopus (95) Google Scholar, 16McNaughton H.J. Thirkettle J.E. Zhang Z.H. Schofield C.J. Jensen S.E. Barton B. Greaves P. Chem. Commun. 1998; : 2325-2326Crossref Scopus (40) Google Scholar), to give deoxyguanidinoproclavaminic acid (17Elson S.W. Baggaley K.H. Davison M. Fulston M. Nicholson N.H. Risbridger G.D. Tyler J.W. J. Chem. Soc.-Chem. Commun. 1993; : 1212-1214Crossref Google Scholar), which is then converted in four steps to (3S,5S)-clavaminic acid. Three of these steps are catalyzed by a single 2-oxoglutarate (2-OG)-dependent oxygenase, clavaminic acid synthase (CAS2) (10Marsh E.N. Chang M.D. Townsend C.A. Biochemistry. 1992; 31: 12648-12657Crossref PubMed Scopus (79) Google Scholar, 18Baldwin J.E. Adlington R.M. Bryans J.S. Bringhen A.O. Coates J.B. Crouch N.P. Lloyd M.D. Schofield C.J. Elson S.W. Baggaley K.H. Cassells R. Nicholson N. J. Chem. Soc.-Chem. Commun. 1990; : 617-619Crossref Google Scholar, 19Elson S.W. Baggaley K.H. Gillett J. Holland S. Nicholson N.H. Sime J.T. Woroniecki S.R. J. Chem. Soc.-Chem. Commun. 1987; : 1736-1738Crossref Scopus (83) Google Scholar, 20Salowe S.P. Marsh E.N. Townsend C.A. Biochemistry. 1990; 29: 6499-6508Crossref PubMed Scopus (124) Google Scholar), and one is catalyzed by proclavaminate amidinohydrolase (PAH) (21Wu T.K. Busby R.W. Houston T.A. McIlwaine D.B. Egan L.A. Townsend C.A. J. Bacteriol. 1995; 177: 3714-3720Crossref PubMed Google Scholar), to give the bicyclic clavam ring system (22Baldwin J.E. Adlington R.M. Bryans J.S. Bringhen A.O. Coates J.B. Crouch N.P. Lloyd M.D. Schofield C.J. Elson S.W. Baggaley K.H. Cassels R. Nicholson N. Tetrahedron. 1991; 47: 4089-4100Crossref Scopus (43) Google Scholar, 23Aidoo K.A. Wong A. Alexander D.C. Rittammer R.A. Jensen S.E. Gene (Amst.). 1994; 147: 41-46Crossref PubMed Scopus (58) Google Scholar, 24Lloyd M.D. Merritt K.D. Lee V. Sewell T.J. Wha-Son B. Baldwin J.E. Schofield C.J. Elson S.W. Baggaley K.H. Nicholson N.H. Tetrahedron. 1999; 55: 10201-10220Crossref Scopus (47) Google Scholar). Crystal structures have been reported for the first four enzymes in the pathway (25Caines M.E. Elkins J.M. Hewitson K.S. Schofield C.J. J. Biol. Chem. 2004; 279: 5685-5692Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 26Miller M.T. Bachmann B.O. Townsend C.A. Rosenzweig A.C. Nat. Struct. Biol. 2001; 8: 684-689Crossref PubMed Scopus (56) Google Scholar, 27Zhang Z. Ren J. Stammers D.K. Baldwin J.E. Harlos K. Schofield C.J. Nat. Struct. Biol. 2000; 7: 127-133Crossref PubMed Scopus (251) Google Scholar, 28Elkins J.M. Clifton I.J. Hernandez H. Doan L.X. Robinson C.V. Schofield C.J. Hewitson K.S. Biochem. J. 2002; 366: 423-434Crossref PubMed Google Scholar) as well as for the gene product of orf6 (OAT2), an ornithine acetyltransferase (29Elkins J.M. Kershaw N.J. Schofield C.J. Biochem. J. 2005; 385: 565-573Crossref PubMed Scopus (26) Google Scholar, 30Kershaw N.J. McNaughton H.J. Hewitson K.S. Hernandez H. Griffin J. Hughes C. Greaves P. Barton B. Robinson C.V. Schofield C.J. Eur. J. Biochem. 2002; 269: 2052-2059Crossref PubMed Scopus (29) Google Scholar) proposed to be involved in the biosynthesis of the l-arginine feedstock for the pathway. The final step in the pathway has also been identified and shown to involve the NADPH-dependent reduction of the labile (3R,5R)-clavaldehyde to give (3R,5R)-clavulanic acid (31Fulston M. Davison M. Elson S.W. Nicholson N.H. Tyler J.W. Woroniecki S.R. J. Chem. Soc.-Perkin Trans. 2001; 1: 1122-1130Crossref Scopus (14) Google Scholar). N2-(2-carboxyethyl)arginine synthase N-glycyl-clavaminic acid synthetase (ORF17) β-lactam synthetase clavaminic acid synthase proclavaminate amidino hydrolase 2-oxoglutarate liquid chromatography-mass spectrometry electrospray ionization MS. N2-(2-carboxyethyl)arginine synthase N-glycyl-clavaminic acid synthetase (ORF17) β-lactam synthetase clavaminic acid synthase proclavaminate amidino hydrolase 2-oxoglutarate liquid chromatography-mass spectrometry electrospray ionization MS. Despite knowledge of the sequences of the likely enzymes involved (4Mellado E. Lorenzana L.M. Rodriguez-Saiz M. Diez B. Liras P. Barredo J.L. Microbiology (Reading). 2002; 148: 1427-1438Crossref PubMed Scopus (56) Google Scholar, 5Li R. Khaleeli N. Townsend C.A. J. Bacteriol. 2000; 182: 4087-4095Crossref PubMed Scopus (50) Google Scholar, 6Hodgson J.E. Fosberry A.P. Rawlinson N.S. Ross H.N. Neal R.J. Arnell J.C. Earl A.J. Lawlor E.J. Gene (Amst.). 1995; 166: 49-55Crossref PubMed Scopus (51) Google Scholar, 23Aidoo K.A. Wong A. Alexander D.C. Rittammer R.A. Jensen S.E. Gene (Amst.). 1994; 147: 41-46Crossref PubMed Scopus (58) Google Scholar, 32Jensen S.E. Paradkar A.S. Mosher R.H. Anders C. Beatty P.H. Brumlik M.J. Griffin A. Barton B. Antimicrob. Agents Chemother. 2004; 48: 192-202Crossref PubMed Scopus (46) Google Scholar), there is little mechanistic information for the apparent double epimerization and oxidative deamination that must occur in the conversion of (3S,5S)-clavaminic acid to (3R,5R)-clavaldehyde. Acylated (3S,5S)-derivatives of clavaminic acid (Fig. 1c), N-acetyl-clavaminic acid, N-acetyl-glycyl-clavaminic acid, and N-glycyl-clavaminic acid have been isolated from a mutant strain of S. clavuligerus deficient in clavulanic acid production (designated dcl8) (33Elson S.W. Gillett J. Nicholson N.H. Tyler J.W. J. Chem. Soc.-Chem. Commun. 1988; : 979-980Crossref Google Scholar). Recently, the latter two of these compounds have been reported in disruption mutants of orf15 and orf16 (32Jensen S.E. Paradkar A.S. Mosher R.H. Anders C. Beatty P.H. Brumlik M.J. Griffin A. Barton B. Antimicrob. Agents Chemother. 2004; 48: 192-202Crossref PubMed Scopus (46) Google Scholar). These compounds have not been observed in disruption mutants of any of the other orfs of the clavulanic acid biosynthetic gene cluster, and the reactions by which they are produced have not been defined. One problem with attempts to define the reactions involved in the biosynthesis of (3R,5R)-clavaldehyde from (3S,5S)-clavaminic acid is the availability of potential substrates with which to challenge candidate enzymes. This problem is common to studies on other biosynthetic pathways leading to many secondary (and some primary) metabolites. Here we have described studies on the latter stages of clavulanic acid biosynthesis in which we approached the problem of a lack of availability of intermediates by utilizing the activity of characterized recombinant enzymes coupled to accessible synthetic intermediates. The results revealed that ORF17 can catalyze the biosynthesis of N-glycyl-clavaminic acid from clavaminic acid in an ATP-dependent manner, supporting the proposal that N-glycyl-clavaminic acid is an intermediate in the clavulanic acid biosynthesis pathway. Materials—Unless otherwise stated, all chemicals were obtained from Sigma. DNA manipulations were carried out by standard protocols (34Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Restriction enzymes were purchased from New England Biolabs Inc. Oligonucleotides were synthesized by SigmaGenosys Ltd. Expression vectors were purchased from Novagen. Protein concentrations were determined by the Bradford method. LC-MS Analyses—The LC-MS system comprised a Waters 600 controller pump with a Waters 2700 sample manager in combination with a Micromass ZMD mass spectrometer using electrospray ionization mass spectrometry (ESI-MS). MassLynx version 3.5 was used for data analysis and processing. The following high performance liquid chromatography columns were used: C18 (150 × 4.60 mm, 5μ) reversephase column (LUNA 5μ C18 (2Baggaley K.H. Brown A.G. Schofield C.J. Nat. Prod. Rep. 1997; 14: 309-333Crossref PubMed Scopus (137) Google Scholar) 100A, Phenomenex). C18 octadecylsilyl guard cartridges (SecurityGuard, Phenomenex) were used throughout and replaced as required. The method was modified from Jensen et al. (32Jensen S.E. Paradkar A.S. Mosher R.H. Anders C. Beatty P.H. Brumlik M.J. Griffin A. Barton B. Antimicrob. Agents Chemother. 2004; 48: 192-202Crossref PubMed Scopus (46) Google Scholar). Samples were eluted with a gradient from 5 to 30% acetonitrile in 100 mm ammonium formate, pH 4.0, at a flow rate of 1 ml·min–1 over 15 min followed by a wash of 30% acetonitrile in 100 mm ammonium formate, pH 4.0, for 10 min before the column was re-equilibrated to 5% acetonitrile in 100 mm ammonium formate, pH 4.0, over 5 min. The production of clavams was also monitored by imidazole-derivatization as described previously (35Foulstone M. Reading C. Antimicrob Agents Chemother. 1982; 22: 753-762Crossref PubMed Scopus (189) Google Scholar), using LC-MS analysis on a LUNA C18 reverse-phase column. Sequence Analysis and Modeling—Homologous polypeptide sequences were obtained from the National Center for Biotechnology Information (NCBI) data base, using the search programs Position Specific Iterated–Basic Local Alignment Search Tool (PSI-BLAST) and Conserved Domain Database (CCD) (36Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59413) Google Scholar). The initial multiple alignments of complete sequences were performed using T-coffee (37Notredame C. Higgins D.G. Heringa J. J. Mol. Biol. 2000; 302: 205-217Crossref PubMed Scopus (5398) Google Scholar) and then manually adjusted to maximize sequence conservation with GeneDoc (38Nicholas K.B. Nicholas Jr., H.B. Deerfield II, D.W. EMBNEW. NEWS. 1997; 4: 14Google Scholar). Structural homologs to ORF17 were obtained using the search program 3D-PSSM (three-dimensional position-specific scoring matrix) (39Kelley L.A. MacCallum R.M. Sternberg M.J.E J. Mol. Biol. 2000; 299: 499-520Crossref PubMed Scopus (1120) Google Scholar). Based on these results, the structure of biotin carboxylase (1DV2) (40Thoden J.B. Blanchard C.Z. Holden H.M. Waldrop G.L. J. Biol. Chem. 2000; 275: 16183-16190Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) was selected to build a sequence-based structural model of ORF17 using MODELLER (41Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10446) Google Scholar). The program Swiss-pdb Viewer (42Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9503) Google Scholar) was used to superimpose biotin carboxylase complexed with ATP (1DV2) (43Fan C. Park I.S. Walsh C.T. Knox J.R. Biochemistry. 1997; 36: 2531-2538Crossref PubMed Scopus (86) Google Scholar) and the ORF17 model. PYMOL was used for visual analysis and rendering of Fig. 2b (44DeLano W.L. Drug Discovery Today. 2005; 10: 213-217Crossref PubMed Scopus (146) Google Scholar). Cloning of the Two Proposed Versions of orf17—Two orf17 sequences have been proposed, differing in length by 92 amino acids. Both the short (32Jensen S.E. Paradkar A.S. Mosher R.H. Anders C. Beatty P.H. Brumlik M.J. Griffin A. Barton B. Antimicrob. Agents Chemother. 2004; 48: 192-202Crossref PubMed Scopus (46) Google Scholar) and the long (4Mellado E. Lorenzana L.M. Rodriguez-Saiz M. Diez B. Liras P. Barredo J.L. Microbiology (Reading). 2002; 148: 1427-1438Crossref PubMed Scopus (56) Google Scholar) forms of the orf17 gene were amplified by PCR from wild-type S. clavuligerus genomic DNA. Both versions were cloned directly into the pET24a(+) vector (Novagen) as an NdeI/BamHI fragment. The primers used for the short version of orf17 were: forward, 5′-ggtggtcatatgaccacacccc-3′; reverse, 5′-ggtggtggatcctcacgactccc-3′. The primers used for the long version of orf17 were: forward, 5′-ggtggtcatatgacccccggggcc-3′; reverse, 5′-ggtggtggatcctcacgactccc-3′. The shorter version of orf17 was also cloned into the pET28a(+) vector as an NdeI/BamHI fragment (primers as above); this produced protein with a thrombin-cleavable N-terminal His tag. It was also cloned, as an NdeI/XhoI fragment, into the pET24a(+) vector (Novagen) to produce a construct encoding for ORF17 with a C-terminal His tag; the primers used are forward, 5′-ggtggtcatatgaccacacccc-3′ and reverse, 5′-ggtggtctcgagcgactccccgccccgctcgcc-3′. Expression and Purification of C-terminal His-tagged ORF17 (Short Form)—Orf17/pET24a(+) (short form) was transformed into Escherichia coli BL21(DE3) competent cells and grown at 37 °C in 2TY medium containing 30 μg·ml–1 kanamycin to an A600 of 0.8. The temperature was reduced to 15 °C, and expression of orf17 was induced by the addition of isopropyl-1-thio-β-d-galactopyranoside at a final concentration of 0.5 mm. Growth was continued for a further 18 h at 15 °C. C-terminal His-tagged ORF17 was purified by sequential use of affinity chromatography (His·Bind® resin, Novagen) and size exclusion chromatography (Superdex S75, Amersham Biosciences) yielding C-terminal His-tagged ORF17 of >95% purity by SDS-PAGE analysis (Fig. 3, lane 4 against SeeBlue® Plus2 pre-stained standard, Invitrogen). Native Molecular Mass Determination—The molecular mass of ORF17 was estimated by size exclusion chromatography (Superdex 200 HR, 10/300). Calibration was carried out using cytochrome C (12 kDa), chymotrypsin (25 kDa), ovalbumin (43 kDa), bovine serum albumin (67 kDa), aldolase (158 kDa), and blue dextran (2000 kDa). An elution volume parameter (Kav) was calculated for each of the calibration proteins, and a calibration curve was constructed. Mass Spectrometric Analyses—For molecular weight determination of ORF17, ESI-MS was performed using a VG Platform II spectrometer with Agilent 1100 series pump and auto-sampler. ORF17 samples were prepared in 1:1 water-acetonitrile containing 0.1% formic acid with a final protein concentration of 10 μm. Soft ESI-MS was preformed on a quadrupole time-of-flight micro with collisional cooling (Micromass UK Ltd., Altrincham, UK). ORF17 samples were prepared in 50 mm ammonium acetate with a final protein concentration of 2 μm. A 20-fold excess of ATP was added to a final protein concentration of 2 μm in 50 mm ammonium acetate. The capillary voltage was set to 3 kV, and the sample cone voltage and extractor cone voltage were changed to 120 and 20 kV, respectively. ORF17 Activity Assays—The rate of ATP hydrolysis was measured at 25 °C by coupling the production of ADP to pyruvate kinase and lactate dehydrogenase while monitoring the loss of NADH at 340 nm (45Cooper R.A. Kornberg H.L. Methods Enzymol. 1969; 13: 309-314Crossref Scopus (38) Google Scholar, 46Penefsky H.S. Bruist M.F. Bergmeyer H.U. Methods of Enzymatic Analysis. 4. John Wiley & Sons, Inc., New York1983: 324-335Google Scholar, 47Allen S.H. Kellermeyer R.W. Stjernholm R.L. Wood H.G. J. Bacteriol. 1964; 87: 171-187Crossref PubMed Google Scholar). The methodology was modified from that reported by Allen et al. (47Allen S.H. Kellermeyer R.W. Stjernholm R.L. Wood H.G. J. Bacteriol. 1964; 87: 171-187Crossref PubMed Google Scholar). The final reaction mixture contained 100 mm Tris-HCl (pH 7.5), 100 mm KCl, 10 mm MgCl2, 1 mm phosphoenolpyruvate, 0.2 mm NADH, varying amounts of ATP (0.1–4.0 mm), 0.7 units of pyruvate kinase, and 1 unit of lactate dehydrogenase (10 μl of stock pyruvate kinase/lactate dehydrogenase from rabbit muscle solution), and the assay was initiated by the addition of 80 μg of ORF17 in a final volume of 1 ml. The release of inorganic phosphate from the hydrolysis of ATP, by ORF17, was confirmed using the EnzChek® phosphate assay kit (Molecular Probes). ORF17 assays were carried out, in the same manner, using the pyruvate kinase/lactate dehydrogenase ATPase assay (45Cooper R.A. Kornberg H.L. Methods Enzymol. 1969; 13: 309-314Crossref Scopus (38) Google Scholar, 46Penefsky H.S. Bruist M.F. Bergmeyer H.U. Methods of Enzymatic Analysis. 4. John Wiley & Sons, Inc., New York1983: 324-335Google Scholar, 47Allen S.H. Kellermeyer R.W. Stjernholm R.L. Wood H.G. J. Bacteriol. 1964; 87: 171-187Crossref PubMed Google Scholar), but reaction mixtures contained 1 mm ATP with 80 μg of ORF17. The assay was initiated with 1 mm potential carboxylate and/or amine substrates in a final volume of 1 ml. Coupled Assay Procedures—Recombinant CEAS, BLS, PAH, and CAS2 were prepared as reported (14Khaleeli N. Li R.F. Townsend C.A. J. Am. Chem. Soc. 1999; 121: 9223-9224Crossref Scopus (81) Google Scholar, 15Bachmann B.O. Li R. Townsend C.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9082-9086Crossref PubMed Scopus (95) Google Scholar, 21Wu T.K. Busby R.W. Houston T.A. McIlwaine D.B. Egan L.A. Townsend C.A. J. Bacteriol. 1995; 177: 3714-3720Crossref PubMed Google Scholar, 24Lloyd M.D. Merritt K.D. Lee V. Sewell T.J. Wha-Son B. Baldwin J.E. Schofield C.J. Elson S.W. Baggaley K.H. Nicholson N.H. Tetrahedron. 1999; 55: 10201-10220Crossref Scopus (47) Google Scholar, 48Busby R.W. Chang M.D.T Busby R.C. Wimp J. Townsend C.A. J. Biol. Chem. 1995; 270: 4262-4269Abstract Full Text PDF PubMed Scopus (43) Google Scholar). A typical coupled enzyme incubation mixture contained 150 mm Tris-HCl (pH 8.0), 12 μm CEAS, BLS, PAH, and CAS2 with appropriate cofactors (30 mm d/l-glyceraldehyde-3-phosphate, 1.5 mm thiamin diphosphate, 10 mm MgCl2, 5 mm ATP, 0.5 mm MnCl2, 10 mm FeSO4, and 10 mm 2-OG) and substrates (30 mm l-arginine or 10 mm deoxyguanidinoproclavaminic acid), in a final volume of 100 μl. The assay mixtures were incubated at 30 °C in a water bath for 1 h, and the reaction was then stopped by heating for 2 min at 100 °C. Protein was removed by centrifugation, and the resulting supernatant was stored at –80 °C before LC-MS analysis on a LUNA C18 reverse-phase column. Controls were conducted in the absence of enzyme(s). Coupled CAS2/ORF17 Activity Assay—The CAS2 assay was modified from that reported by Lloyd et al. (24Lloyd M.D. Merritt K.D. Lee V. Sewell T.J. Wha-Son B. Baldwin J.E. Schofield C.J. Elson S.W. Baggaley K.H. Nicholson N.H. Tetrahedron. 1999; 55: 10201-10220Crossref Scopus (47) Google Scholar). The final incubation mixture contained 100 mm Tris-HCl (pH 7.5), 2 mm dithiothreitol, 10 mm 2-OG, 2 mm FeSO4, 50 μg of CAS2, and 2.5 mm synthetic racemic proclavaminic acid (24Lloyd M.D. Merritt K.D. Lee V. Sewell T.J. Wha-Son B. Baldwin J.E. Schofield C.J. Elson S.W. Baggaley K.H. Nicholson N.H. Tetrahedron. 1999; 55: 10201-10220Crossref Scopus (47) Google Scholar) in a final volume of 50 μl. The assay mixture was incubated at 37 °C in a water bath. After 20 min, more FeSO4 and dithiothreitol (10 μl of a 10 mm stock of each) were added. After 40 min, the reaction mixture was centrifuged at 14,000 rpm for 2 min, to remove any precipitate, and the supernatant was filtered through a 10-kDa molecular mass cut-off membrane and centrifuged at 14,000 rpm for 10 min. 25 μl of the CAS2-free filtrate was then combined with a buffer solution of 10 mm MgCl2, 100 mm KCl, and 100 mm Tris-HCl, pH 7.5, containing 2 mm ATP, 2 mm glycine, or alternative potential substrates, and the assay was initiated by the addition of 80 μg of ORF17 in a final volume of 50 μl. The mixture was incubated at 37 °C in a water bath for 30 min before the reaction was stopped by the addition of acetone (120 μl). After centrifugation, the acetone was removed in vacuo (using a Speed-Vac SC110, Savant), and the mixture was then analyzed by LC-MS. This assay and substrate specificity assays were also conducted using the pyruvate kinase/lactate dehydrogenase ATPase assay (45Cooper R.A. Kornberg H.L. Methods Enzymol. 1969; 13: 309-314Crossref Scopus (38) Google Scholar, 46Penefsky H.S. Bruist M.F. Bergmeyer H.U. Methods of Enzymatic Analysis. 4. John Wiley & Sons, Inc., New York1983: 324-335Google Scholar, 47Allen S.H. Kellermeyer R.W. Stjernholm R.L. Wood H.G. J. Bacteriol. 1964; 87: 171-187Crossref PubMed Google Scholar), using either 25 μl of clavaminic acid stock, produced from CAS2 assays (as above), or alternative potential substrates. The assay was initiated with 1 mm potential carboxylate substrates in a final volume of 1 ml. Expression and Purification of ORF17—Two possible start codons have been proposed for orf17 (4Mellado E. Lorenzana L.M. Rodriguez-Saiz M. Diez B. Liras P. Barredo J.L. Microbiology (Reading). 2002; 148: 1427-1438Crossref PubMed Scopus (56) Google Scholar, 32Jensen S.E. Paradkar A.S. Mosher R.H. Anders C. Beatty P.H. Brumlik M.J. Griffin A. Barton B. Antimicrob. Agents Chemother. 2004; 48: 192-202Crossref PubMed Scopus (46) Google Scholar). The sequence of orf17 identified by Mellado et al. (4Mellado E. Lorenzana L.M. Rodriguez-Saiz M. Diez B. Liras P. Barredo J.L. Microbiology (Reading). 2002; 148: 1427-1438Crossref PubMed Scopus (56) Google Scholar) encodes a protein of 529 amino acids, whereas that proposed by Jensen et al. (32Jensen S.E. Paradkar A.S. Mosher R.H. Anders C. Beatty P.H. Brumlik M.J. Griffin A. Barton B. Antimicrob. Agents Chemother. 2004; 48: 192-202Crossref PubMed Scopus (46) Google Scholar) lacks the first 92 residues (this difference is reflected in the different numbering schemes for each gene product). There are two further differences in the predicted polypeptide sequences (highlighted by bold italicized residues), 473VEKGVKLLKR482 compared with 381VEKGDKLLQR390, for Mellado et al. (4Mellado E. Lorenzana L.M. Rodriguez-Saiz M. Diez B. Liras P. Barredo J.L. Microbiology (Reading). 2002; 148: 1427-1438Crossref PubMed Scopus (56) Google Scholar) and Jensen et al. (32J" @default.
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• W2055374672 title "ORF17 from the Clavulanic Acid Biosynthesis Gene Cluster Catalyzes the ATP-dependent Formation of N-Glycyl-clavaminic Acid" @default.
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