Isolation of dihydroclavaminic acid, an intermediate in the biosynthesis of clavulanic acid

Baldwin, Jack E.; Adlington, Robert M.; Bryans, Justin S.; Bringhen, Alain O.; Coates, J. B.; Crouch, Nicholas P.; Lloyd, Matthew D.; Schofield, Christopher J.; Elson, Stephen W.; Baggaley, Keith H.; Cassels, Robert; Nicholson, Neville

doi:10.1016/s0040-4020(01)86446-2

Cited by 51 publications

(19 citation statements)

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“…There was no evidence for the production of N-glycyl-proclavaminic acid or N-glycyl-dihydroclavaminic acid upon incubation of ORF17, ATP, and glycine with the crude products of the CAS assay. Separation of dihydroclavaminic acid and clavaminic acid on a preparative scale to allow each component to be assayed separately was not possible, due to the small amounts of unstable dihydroclavaminic acid available (22,24). Thus, the CAS2 incubation mixture was filtered through a 10-kDa molecular mass cut off membrane to remove CAS2, and the filtrate was then incubated with ORF17, ATP, and glycine (Fig.…”

Section: And N-␣-acetyl-l-lysinementioning

confidence: 99%

“…␤-Lactam formation is catalyzed by ␤-lactam synthetase (BLS) (15,16), to give deoxyguanidinoproclavaminic acid (17), 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) (10, 18 -20), and one is catalyzed by proclavaminate amidinohydrolase (PAH) (21), to give the bicyclic clavam ring system (22)(23)(24). Crystal structures have been reported for the first four enzymes in the pathway (25)(26)(27)(28) as well as for the gene product of orf6 (OAT2), an ornithine acetyltransferase (29,30) proposed to be involved in the biosynthesis of the L-arginine feedstock for the pathway.…”

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confidence: 99%

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ORF17 from the Clavulanic Acid Biosynthesis Gene Cluster Catalyzes the ATP-dependent Formation of N-Glycyl-clavaminic Acid

Arulanantham

Kershaw

Hewitson

et al. 2006

Journal of Biological Chemistry

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Section: And N-␣-acetyl-l-lysinementioning

confidence: 99%

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confidence: 99%

ORF17 from the Clavulanic Acid Biosynthesis Gene Cluster Catalyzes the ATP-dependent Formation of N-Glycyl-clavaminic Acid

Arulanantham

Kershaw

Hewitson

et al. 2006

Journal of Biological Chemistry

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“…Guanidino proclavaminic acid is hydrolyzed to give proclavaminic acid by the action of proclavaminate amidinohydrolase (PAH) (46), which is encoded by pah1 and pah2 (1,15,18,46). Next, in a series of two sequential reactions, CAS converts proclavaminic acid to clavaminic acid (4,36). Clavaminic acid is thought to be the branch point of the pathway leading to clavulanic acid and the 5S clavams (10).…”

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The Paralogous Pairs of Genes Involved in Clavulanic Acid and Clavam Metabolite Biosynthesis Are Differently Regulated in Streptomyces clavuligerus

2004

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Carboxyethylarginine synthase, encoded by the paralogous ceaS1 and ceaS2 genes, catalyzes the first reaction in the shared biosynthetic pathway leading to clavulanic acid and the other clavam metabolites in Streptomyces clavuligerus. The nutritional regulation of ceaS1 and ceaS2 expression was analyzed by reverse transcriptase PCR and by the use of the enhanced green fluorescent protein-encoding gene (egfp) as a reporter. ceaS1 was transcribed in complex soy medium only, whereas ceaS2 was transcribed in both soy and defined starchasparagine (SA) media. The transcriptional start points of the two genes were also mapped to a C residue 98 bp upstream of ceaS1 and a G residue 51 bp upstream of the ceaS2 start codon by S1 nuclease protection and primer extension analyses. Furthermore, transcriptional mapping of the genes encoding the beta-lactam synthetase (bls1) and proclavaminate amidinohydrolase (pah1) isoenzymes from the paralogue gene cluster indicated that a single polycistronic transcript of ϳ4.9 kb includes ceaS1, bls1, and pah1. The expression of ceaS1 and ceaS2 in a mutant strain defective in the regulatory protein CcaR was also examined. ceaS1 transcription was not affected in the ccaR mutant, whereas that of ceaS2 was greatly reduced compared to the wild-type strain. Overall, our results suggest that different mechanisms are involved in regulating the expression of ceaS1 and ceaS2, and presumably also of other paralogous genes that encode proteins involved in the early stages of clavulanic acid and clavam metabolite biosynthesis.

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“…1, compound 4) (1,65), and to govern successive oxidative cyclization and desaturation to clavaminic acid ( Fig. 1, compound 5) (13,18,23,54).…”

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Expansion of the Clavulanic Acid Gene Cluster: Identification and In Vivo Functional Analysis of Three New Genes Required for Biosynthesis of Clavulanic Acid by Streptomyces clavuligerus

Khaleeli

Townsend

2000

J Bacteriol

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Isolation of dihydroclavaminic acid, an intermediate in the biosynthesis of clavulanic acid

Cited by 51 publications

References 14 publications

ORF17 from the Clavulanic Acid Biosynthesis Gene Cluster Catalyzes the ATP-dependent Formation of N-Glycyl-clavaminic Acid

ORF17 from the Clavulanic Acid Biosynthesis Gene Cluster Catalyzes the ATP-dependent Formation of N-Glycyl-clavaminic Acid

The Paralogous Pairs of Genes Involved in Clavulanic Acid and Clavam Metabolite Biosynthesis Are Differently Regulated in Streptomyces clavuligerus

Expansion of the Clavulanic Acid Gene Cluster: Identification and In Vivo Functional Analysis of Three New Genes Required for Biosynthesis of Clavulanic Acid by Streptomyces clavuligerus

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