Stephen W. Elson

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

Abstract A primary isotope effect was utilised in an in vitro study to allow the isolation and characterisation of an intermediate between proclavaminic acid and clavaminic acid, in clavulanic acid biosynthesis. 1

Evidence that arginine is a later metabolic intermediate than ornithine in the biosynthesis of clavulanic acid by Streptomyces clavuligerus

The feeding of radiolabelled ornithine and arginine to mutants of Streptomyces clavuligerus auxotrophic for arginine has shown that ornithine has to be converted to arginine prior to incorporation into clavulanic acid.

Isolation of two novel intracellular β-lactams and a novel dioxygenase cyclising enzyme from Streptomyces clavuligerus

Two novel β-lactams, one monocyclic, the other bicyclic, and a dioxygenase enzyme which converts the former to the latter, have been isolated from the mycelium of the clavulanic acid producing organism Streptomyces clavuligerus ATCC 27064.

The identification of three new biosynthetic intermediates and one further biosynthetic enzyme in the clavulanic acid pathway

Labelling experiments are described that identify three new compounds, N2-(2-carboxyethyl)arginine, 5-guanidino-(2-oxoazetidin-1-yl)pentanoic acid, and 3-hydroxy-5-guanidino-2-(2-oxoazetidin-1-yl)pentanoic acid as biosynthetic precursors of proclavaminic acid and hence clavulanic acid in Streptomyces clavuligerus TCC 27064 and a new amidino hydrolase, which hydrolyses 3-hydroxy-5-guanidino-2-(2-oxoazetidin-1-yl)pentanoic acid to proclavaminic acid has been characterised.

Evidence that the immediate biosynthetic precursor of clavulanic acid is its N-aldehyde analogue

(3R, 5R) Clavulanate-9-aldehyde 1 has been detected in Streptomyces clavuligerus and an NADPH dependent dehydrogenase capable of reducing 1 to clavulanic acid 2 has been isolated from this organism.

Expression in Escherichia coli of a clavaminic acid synthase isozyme : a trifunctional oxygenase involved in clavulanic acid biosynthesis

Abstract Purification and sequencing of clavaminic acid synthase (CAS) activities from S. clavuligerus SC2 provided evidence for the existence of two CAS isozymes. One of these isozymes was cloned and expressed in E. coli. The recombinant CAS isozyme was shown to catalyse the hydroxylation of (2S)-5-guanidino-2-(2′-oxoazetidin-1′-yl)pentanoic acid, in addition to the production of clavaminic acid from dihydroclavaminic acid via proclavaminic acid, consistent with the trifunctional role proposed for clavaminic acid synthase in the biosynthesis of clavulanic acid. A preliminary purification and characterisation of recombinant isozyme is reported.

A substrate analogue study on clavaminic acid synthase: possible clues to the biosynthetic origin of proclavamic acid

Incubation of (2S)-5-amino-(2′-oxoazetidin-1′-yl)pentanoic acid with clavaminic acid synthase gave (2S)-5-amino-2-(2′-oxoazetidin-1′-yl)pent-3,4-enoic acid as the major product, together with a small amount of proclayaminic acid; modification of the amino group to the guanyl group biased the mode of reactivity from desaturation to hydroxylation.

N-acyl derivatives of clavaminic acid produced by a mutant of Streptomyces clavuligerus

A mutant of Streptomyces clavuligerus, blocked in clavulanic acid production, was found to accumulate three N-acyl derivatives of the β-lactam clavaminic acid in the culture broth.

Clavulanic acid biosynthesis; the final steps

The chemically unstable anabolite (3R,5R)-clavulanate-9-aldehyde 1 and an NADPH dependent dehydrogenase have been detected in the broth of Streptomyces clavuligerus. The purified enzyme was shown to make clavulanic acid by reduction of the aldehydic moiety of synthetic 1 to the allylic alcohol of clavulanic acid 2. A DNA sequence corresponding to the enzyme’s N-terminal amino acid sequence was located within the clavulanic acid biosynthetic gene cluster. We have named this novel enzyme clavulanic acid dehydrogenase (CAD). In an attempt to determine the origin of 1, fermentations of S. clavuligerus were fed ornithine labelled with stable isotopes in the carboxy group. The results of these experiments are discussed.

Two novel arginine derivatives from a mutant of Streptomyces clavuligerus

Two novel arginine derivatives, N2-(2-carboxyethyl)arginine and N2-(2-carboxyethyl)-3-hydroxyarginine, are produced by a mutant of Streptomyces clavuligerus dclH 65, which is blocked in clavulanic acid biosynthesis, and the structures of the compounds indicate that they may be involved in clavulanic acid biosynthesis.