2IHT

Thiamine-pyrophosphate dependent N(2)-(2-carboxyethyl)arginine synthase catalyses the initial step in the biosynthesis of the clinically relevant beta-lactamase inhibitor, clavulanic acid. The reaction involves the condensation of two primary metabolites, D-glyceraldehyde-3-phosphate and l-arginine to form N(2)-(2-carboxyethyl)arginine and phosphate. This C-N bond forming reaction forms an unusual alpha, beta-unsaturated acyl-thiamine-pyrophosphate intermediate. This enzyme is the first gene in the clavulanic acid cluster of the organism Streptomyces clavuligerus, with 18 other genes of the genome known to take part in this biosynthetic pathway. Besides this enzyme’s biosynthetic importance, it is also of mechanistic interest because of its unusual mechanism for a thiamine-pyrophosphate dependent enzyme.

As expected of a thiamine-pyrophosphate dependent enzyme, sequence analysis indicates the presence of a thiamine binding mode – a motif well conserved between enzymes which use this cofactor. The mechanism can require thiamine as a cofactor in two ways: i) to effect the nucleophilic attack at a carbonyl group, and ii) to stabilise the carbanion formed after proton transfer (or decarboxylation, as catalysed by some thiamine-pyrophosphate utilising enzymes). The enzyme is tetrameric, both physiologically and in crystallography data.