Biotin synthase from Escherichia coli: isolation of an enzyme-generated intermediate and stoichiometry of S-adenosylmethionine use

• Published in: Biochemical journal Vol. 330 ( Pt 3); no. 3; pp. 1079 - 1085
• Main Authors: Shaw, N M, Birch, O M, Tinschert, A, Venetz, V, Dietrich, R, Savoy, L A
• Format: Journal Article
• Language: English
• Published: England 15.03.1998
• Subjects: Chromatography, High Pressure Liquid; Cloning, Molecular; Cystine - metabolism; Escherichia coli - metabolism; Hydrogen-Ion Concentration; Kinetics; Models, Chemical; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; S-Adenosylmethionine - metabolism; Sulfurtransferases - biosynthesis; Sulfurtransferases - isolation & purification; Sulfurtransferases - metabolism
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3301079

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 synthase reaction. The pH optimum was between 8 and 8.5, and the reaction velocity 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. the enzyme was not acting catalytically. HPLC analysis of reaction mixtures revealed the presence of a compound with the characteristics of an intermediate: (1) it was labelled with 14C, and therefore derived from the [14C]dethiobiotin substrate; (2) it was present only in reaction mixtures containing biotin synthase; (3) it was not derived from [14C]biotin; (4) 35S from [35S]cystine was incorporated into the intermediate during the reaction; (5) its synthesis was dependent on the presence of S-adenosylmethionine, and was decreased when free cysteine was omitted from the reaction; (6) it could be isolated from the reaction mixture by chromatography and then re-introduced into an assay as the substrate, whereupon it was converted to biotin; (7) this conversion to biotin was S-adenosylmethionine-dependent. During the reaction S-adenosylmethionine was cleaved to methionine and presumably 5'-deoxyadenosine. Observation of the intermediate allowed us to perform experiments to determine the stoichiometry of S-adenosylmethionine use. We propose that two molecules of S-adenosylmethionine are used to synthesize one molecule of biotin, i.e. one from dethiobiotin to the intermediate, and a second from the intermediate to biotin.