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 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.
Shaw
N M
NM
Biotechnology Department, Lonza AG, CH-3930 Visp, Switzerland.
Birch
O M
OM
Tinschert
A
A
Venetz
V
V
Dietrich
R
R
Savoy
L A
LA
D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003553:Cystine / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism*; D013466:Sulfurtransferases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D003553:Cystine; D012436:S-Adenosylmethionine; D013466:Sulfurtransferases; C057985:biotin synthetase