Biotin synthase mechanism: mutagenesis of the YNHNLD conserved motif.
Biotin synthase, a member of the "radical SAM" family, catalyzes the final step of the biotin biosynthetic pathway, namely, the insertion of a sulfur atom into dethiobiotin (DTB). The active form of the enzyme contains two iron-sulfur clusters, a [4Fe-4S](2+) cluster liganded by Cys-53, Cys-57, and Cys-60 and the S-adenosylmethionine (AdoMet or SAM) cosubstrate and a [2Fe-2S](2+) cluster liganded by Cys-97, Cys-128, Cys-188, and Arg-260. Single-point mutation of each of these six conserved cysteines produced inactive variants. In this work, mutants of other highly conserved residues from the Y(150)NHNLD motif are described. They have properties similar to those of the wild-type enzyme with respect to their cluster content and characteristics. For all of them, the as-isolated form, which contains an air-stable [2Fe-2S](2+) center, can additionally accommodate an air-sensitive [4Fe-4S](2+) center which is generated by incubation under anaerobic conditions with Fe(2+) and S(2-). Their spectroscopic properties are similar to those of the wild type. However, they are inactive, except the mutant H152A that exhibits a weak activity. We show that the mutants, inactive in producing biotin, are also unable to cleave AdoMet and to produce the deoxyadenosyl radical (AdoCH(2)(*)). In the case of H152A, a value of 5.5 +/- 0.4 is found for the 5'-deoxyadenosine (AdoCH(3)):biotin ratio, much higher than the value of 2.8 +/- 0.3 usually observed with the wild type. This reveals a greater contribution of the abortive process in which the AdoCH(2)(*) radical is quenched by hydrogen atoms from the protein or from some components of the system. Thus, in this case, the coupling between the production of AdoCH(2)(*) and its reaction with the hydrogen at C-6 and C-9 of DTB is less efficient than that in the wild type, probably because of geometry's perturbation within the active site.
Lotierzo
Manuela
M
Université Pierre et Marie Curie-Paris 6, CNRS UMR 7613, (Synthèse, Structure et Fonction de Molécules Bioactives), FR2769, Case Courrier 182, 4 place Jussieu, 75005 Paris, France.
Raux
Evelyne
E
Tse Sum Bui
Bernadette
B
Goasdoue
Nicole
N
Libot
Francine
F
Florentin
Dominique
D
Warren
Martin J
MJ
Marquet
Andrée
A
D020816:Amino Acid Motifs / Q000235:genetics*; D000595:Amino Acid Sequence; D017124:Conserved Sequence / Q000235:genetics*; D003839:Deoxyadenosines / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet; D013466:Sulfurtransferases / Q000235:genetics* / Q000378:metabolism
D003839:Deoxyadenosines; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; C033714:5'-deoxyadenosine; D013466:Sulfurtransferases; C087072:biotin synthase, E coli