Reductive cleavage of S-adenosylmethionine by biotin synthase from Escherichia coli.
Biotin synthase (BioB) catalyzes the insertion of a sulfur atom between the C6 and C9 carbons of dethiobiotin. Reconstituted BioB from Escherichia coli contains a [4Fe-4S](2+/1+) cluster thought to be involved in the reduction and cleavage of S-adenosylmethionine (AdoMet), generating methionine and the reactive 5'-deoxyadenosyl radical responsible for dethiobiotin H-abstraction. Using EPR and Mössbauer spectroscopy as well as methionine quantitation we demonstrate that the reduced S = 1/2 [4Fe-4S](1+) cluster is indeed capable of injecting one electron into AdoMet, generating one equivalent of both methionine and S = 0 [4Fe-4S](2+) cluster. Dethiobiotin is not required for the reaction. Using site-directed mutagenesis we show also that, among the eight cysteines of BioB, only three (Cys-53, Cys-57, Cys-60) are essential for AdoMet reductive cleavage, suggesting that these cysteines are involved in chelation of the [4Fe-4S](2+/1+) cluster.
Ollagnier-de Choudens
Sandrine
S
Laboratoire de Chimie et Biochimie des Centres Rédox Biologiques, Departement de Biologie Moléculaire et Structurale-Chimie Biologie, CEA/CNRS/Université Joseph Fourier, UMR 5047, Grenoble, France.
Sanakis
Yiannis
Y
Hewitson
Kirsty S
KS
Roach
Peter
P
Münck
Eckard
E
Fontecave
Marc
M
D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008715:Methionine / Q000737:chemistry; D008956:Models, Chemical; D009154:Mutation; D012436:S-Adenosylmethionine / Q000737:chemistry* / Q000494:pharmacology*; D015204:Spectroscopy, Mossbauer; D013466:Sulfurtransferases / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
D001710:Biotin; C004749:desthiobiotin; D012436:S-Adenosylmethionine; D008715:Methionine; D013466:Sulfurtransferases; C057985:biotin synthetase; D003545:Cysteine