Keith H. Baggaley

PRODUCT-SUBSTRATE ENGINEERING BY BACTERIA : STUDIES ON CLAVAMINATE SYNTHASE, A TRIFUNCTIONAL DIOXYGENASE

Evidence is presented that clavaminate synthase (CS) catalyses three oxidative reactions in the clavulanic acid biosynthetic pathway. The first CS catalysed step (hydroxylation) is separated from the latter two (oxidative cyclisation and desaturation) by the action of a hydrolytic enzyme, proclavaminate amidinohydrolase, which modifies (or ‘mutates’) the sidechain of the product of the first reaction thereby converting it into a substrate for the second CS catalysed reaction.

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.

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.

Chemo-enzymatic synthesis of bicyclic γ-lactams using clavaminic acid synthase

Abstract Incubation of the γ-lactam analogue of proclavaminic acid, (±)-threo-5-amino-3-hydroxy-2-(1′-aza-2′-oxocyclopentyl)-pentanoic acid, led to production of two bicyclic γ-lactam products.

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.

Studies on the stereospecificity of the clavaminic acid synthase catalysed hydroxylation reaction

Incubation of (2S,3S)-5-guanidino[2,3-2H2]-2-(2′-oxoazetidin-1′-yl)pentanoic acid and (2S,3R)-5-guanidino-[3-2H1]-2-(2′-oxoazetidin-1′-yl)pentanoic acid with clavaminic acid synthase resulted in highly stereospecific hydroxylation at C-3, with removal of the pro-R hydrogen or deuterium, respectively.

Investigation of the stereospecificity of clavaminic acid synthase in the desaturation of dihydroclavaminic acid to clavaminic acid

Incubations of (4R)- and (4S)-[4-2H1]-proclavaminic acid with clavaminic acid synthase resulted in the stereospecific removal of the deuterium and hydrogen respectively from C-4, in their conversions to clavaminic acid, suggesting an enzyme catalysed syn-eliminaion for the desaturation of dihydroclavaminic acid to clavaminic acid.

Substrate analogue studies on clavaminic acid synthase

Incubation of N-α-acetyl-L-ornithine with clavaminic acid synthase led to the isolation of (E)-(2S)-5-amino-2-acetamidopent-3-enoic acid and (2S)-2-acetamido-5-amino-3-hydroxypentanoic acid, while incubation of N-α-acetyl-L-arginine led to the isolation of (2S)-2-acetamido-5-guanidino-3-hydroxypentanoic acid.