Nicholas P. Crouch

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

A mechanistic rationalisation for the substrate specificity of recombinant mammalian 4-hydroxyphenylpyruvate dioxygenase (4-HPPD)

Abstract The isolation and purification of α-ketoisocaproate dioxygenase [α-KICD] from rat liver is described. Sequence determination of the purified protein revealed it to have complete homology to rat liver 4-hydroxyphenyl-pyruvate dioxygenase [4-HPPD] which was confirmed by the cloning and expression of the gene encoding 4-HPPD in E. coli. Examination of the substrate specificity of the resulting soluble recombinant protein revealed it to be capable of the oxidative decarboxylation of a range of ketoacids derived from proteinogenic amino acids. The significance of the turnover of these different ketoacids is discussed in relation to the mechanism of this fascinating enzyme.

Cephalosporin biosynthesis: A branched pathway sensitive to an isotope effect

Abstract Incubation of penicillin N ( 3a ) with partially purified deacetoxy/deacetylcephalosporin C synthase (DAOC/DAC synthase) from Cephalosporium acremonium CO 728 gave in addition to the expected products, deacetoxycephalosporin C and deacetylcephalosporin C, a third β-lactam metabolite as a 3β-hydroxy-3α-methylcepham ( 9a ). Production of the 3β-hydroxycepham was promoted from [3- 2 H]penicillin N ( 3b ) which was rationalised by the operation of a kinetic isotope effect on a branched pathway in the enzymic process. The oxygen of the 3β-hydroxy group was shown to be derived in part from molecular oxygen. In addition, the 2β-methyl group of penicillin N was shown to be incorporated into C2 of the 3β-hydroxy-3α-methylcepham, a result in stereochemical accord with the equivalent transformation of the 2β-methyl group of penicillin N into C2 of deacetoxycephalosporin C 1 . A mechanistic interpretation, consistent with these observations, is offered.

An expedient synthesis of trisubstituted allenes

Abstract Di-α-alkylated-α-(β-keto sulfone) alkynes provide a facile entry to trisubstituted allenes upon treatment with aluminium amalgam.

Incorporation of 18O-labelled water into oxygenated products produced by the enzyme deacetoxy/deacetylcephalosporin C synthase

Abstract 18O-labelling experiments have been conducted with the enzyme deacetoxy/deacetylcephalosporin C synthase. Incubations of [2-13C,3-2H]penicillin N and [4-2H]exomethylene cephalosporin C were carried out with 18O2 or H218O, and 18O-incorporation from both label sources was observed into the oxygenated enzymic products. This is believed to be the first example of H218O incorporation into products of an α-ketoglutarate dependent dioxygenase. An oxygen-exchange process at an enzyme-bound intermediate is proposed to account for this incorporation.

The C-terminal of rat 4-hydroxyphenylpyruvate dioxygenase is indispensable for enzyme activity.

We have cloned and overexpressed rat 4‐hydroxyphenylpyruvate dioxygenase (4HPPD) in Escherichia coli. The soluble, active recombinant enzyme was shown to contain both 4HPPD and α‐ketoisocaproate dioxygenase (αKICD) activity. However, upon truncation of the 14 amino acids at the C‐terminus by site‐directed mutagenesis, the resulting mutant enzyme (rat F antigen) exhibited complete loss of 4HPPD and αKICD activities. This finding suggests that the C‐terminal extension domain plays an essential role in the catalytic activity of the enzyme.

4-Hydroxyphenylpyruvate dioxygenase appears to display α-ketoisocaproate dioxygenase activity in rat liver

Abstract Isolation and sequence determination of a protein exhibiting α-ketoisocaproate dioxygenase activity from rat liver revealed that the protein was identical to the liver-specific rat F antigen which is believed to be a species variant of 4-hydroxyphenylpyruvate dioxygenase. Isolation and sequence determination of a protein exhibiting α-ketoisocaproate dioxygenase activity from rat liver revealed that the protein was identical to the liver-specific rat F antigen which is believed to be a species variant of 4-hydroxyphenylpyruvate dioxygenase.

The enzymatic conversion of exomethylene cephalosporin c into deacetyl cephalosporin c and the role of molecular oxygen in cephalosporin c biosynthesis

Abstract Cell free extracts from Cephalosporium acremonium containing deaoetoxy/-deacetyl cephalosporin C Synthetase activities have been shown to convert Exomethylene cephalosporin C directly to deacetyl cephalosporin C. Labelling experiments in the presence of 13 O 2 , indicated the source of the 3-methylene-oxygen of the latter was dioxygen. 1

Substrate specificity of recombinant Streptomyces clavuligerus deacetoxycephalosporin C synthase

Abstract The soluble, high-level expression in Escherichia coli of the deacetoxycephalosporin C synthase gene from Streptomyces clavuligerus and the substrate specificity of the recombinant enzyme are described.

INITIAL STUDIES ON THE SUBSTRATE SPECIFICITY OF SOLUBLE RECOMBINANT 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE FROM RAT LIVER

The molecular cloning and expression of the cDNA encoding 4-hydroxyphenylpyruvate dioxygenase (rat F antigen) into E.coli and initial studies concerning the substrate specificity of the recombinant protein are described.