BIOTIN SYNTHASE FROM ESCHERICHIA-COLI - ISOLATION OF AN ENZYME-GENERATED INTERMEDIATE AND STOICHIOMETRY OF S-ADENOSYLMETHIONINE USE

A cell-free extract from Escherichia coli containing an E. coli biotin synthase that was expressed to approx. 1% of soluble cell protein by cloning the E. coli bioB gene was used to investigate the biotin synth ase reaction. The pH optimum was between 8 and 8.5, and the reaction v elocity was dependent on the concentrations of dethiobiotin, cysteine, S-adenosylmethionine and asparagine. The catalytic-centre activity of the enzyme in vitro was estimated to be 0.95 h(-1), and each molecule of enzyme turned over less than one molecule of dethiobiotin. i.e. th e enzyme was not acting catalytically. HPLC analysis of reaction mixtu res revealed the presence of a compound with the characteristics of an intermediate: (1) it was labelled with C-14, and therefore derived fr om the [C-14]dethiobiotin substrate; (2) it was present only in reacti on mixtures containing biotin synthase; (3) it was not derived from [C -14]biotin; (4) S-35 from [S-35]cystine was incorporated into the inte rmediate during the reaction; (5) its synthesis was dependent on the p resence of S-adenosylmethionine, and was decreased when free cysteine was omitted from the reaction; (6) it could be isolated from the react ion mixture by chromatography and then re-introduced into an assay as the substrate, whereupon it was converted to biotin; (7) this conversi on to biotin was S-adenosylmethionine-dependent. During the reaction S -adenosylmethionine was cleaved to methionine and presumably 5'-deoxya denosine. Observation of the intermediate allowed us to perform experi ments to determine the stoichiometry of S-adenosylmethionine use. We p ropose that two molecules of S-adenosylmethionine are used to synthesi ze one molecule of biotin, i.e. one from dethiobiotin to the intermedi ate, and a second from the intermediate to biotin.