9-Mercaptodethiobiotin Is Formed as a Competent Catalytic Intermediate by Escherichia coli Biotin Synthase

Abstract

Biotin synthase (BS) catalyzes the oxidative addition of a sulfur atom to dethiobiotin (DTB) to generate the biotin thiophane ring. This enzyme is an S-adenosylmethionine (AdoMet) radical enzyme that catalyzes the reductive cleavage of AdoMet, generating methionine and a transient 5´-deoxyadenosyl radical. In our working mechanism, the 5´-deoxyadenosyl radical oxidizes DTB by abstracting a hydrogen from C6 or C9, generating a dethiobiotinyl carbon radical that is quenched by sulfide from a [2Fe-2S]2+ cluster. A similar reaction sequence directed at the other position generates the second C-S bond in the thiophane ring. Since the BS active site holds only one AdoMet and one DTB, it follows that dissociation of methionine and 5´-deoxyadenosine and binding of a second equivalent of AdoMet must be intermediate steps in the formation of biotin. During these dissociation/association steps, a discrete DTB-derived intermediate must remain bound to the enzyme. In the present work, we confirm that the conversion of DTB to biotin is accompanied by the reductive cleavage of 2 equivalents of AdoMet. A discrepancy between DTB consumption and biotin formation suggests the presence of an intermediate, and we use LCMS to demonstrate that this intermediate is indeed 9-mercaptodethiobiotin, generated at ~10% of the total enzyme concentration. The amount of intermediate observed is increased when the reaction is run with substoichiometric levels of AdoMet or with defective enzyme containing the mutation Asn153Ser. The retention of 9-mercaptodethiobiotin as a tightly-bound intermediate is consistent with a mechanism involving the stepwise radical-mediated oxidative abstraction of sulfide from an iron-sulfur cluster.

Biotin and Lipoic Acid: Synthesis, Attachment, and Regulation

“…The SAM is positioned such that reductive cleavage by the [4Fe-4S] cluster could readily occur while the DTB is positioned such that the C-9 carbon can accept a sulfur atom from the [2Fe-2S] cluster (58). Indeed, 9-mercaptodethiobiotin is a catalytic intermediate (60)(61)(62) This latter finding fits with the belief of most workers in the field that the [2Fe-2S] cluster is the immediate source of the biotin sulfur atom. This belief is supported by experiments in which each of the sulfurcontaining small molecules of the defined in vitro reaction mixture was labeled with 35 S and incorporation of the isotope into biotin was measured (see ref.…”

Catalytic Activity of the Anaerobic Tyrosine Lyase Required for Thiamine Biosynthesis in Escherichia coli

“…The ratelimiting step for ThiH reaction catalysis is unknown, but for anaerobic sulfatase-maturating enzyme, another member of the radical AdoMet family, the observed deuterium isotope effect indicates that hydrogen atom abstraction by the 5Ј-deoxyadenosyl radical is the rate-limiting step (28). The burst phase rate constant obtained for ThiH reaction catalysis (53 Ϯ 6 ϫ 10 Ϫ4 s Ϫ1 ) is comparable with other members of the radical AdoMet family; for example, apparent rate constants have been reported as 60 ϫ 10 Ϫ4 and 12 ϫ 10 Ϫ4 s Ϫ1 for the AtsB (formylglycine-generating enzyme) (29) and biotin synthase (30), respectively.…”

Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions

“…The function of the auxiliary cluster has not been fully elucidated, but studies with 34 S labeled LipA suggest both of the sulfur atoms are transferred from a single LipA molecule [16], which may indicate the auxiliary cluster is functioning as the sulfur donor. The high degree of sequence similarity between biotin synthase and lipoyl synthase [15] which are both required for transformations resulting in sulfur insertion [17] suggests they may function by related mechanisms and kinetic [18], structural [19] and spectroscopic data [20,21] indicate that the auxiliary [2Fe-2S] cluster of BioB functions is the sulfur donor for biotin formation. A further subgroup of the RS superfamily, the methylthiotransferases, catalyze the insertion of thiomethyl groups into C-H bonds of proteins [22,23] and nucleic acids [24][25][26], but these enzymes can use exogenously added sulfide as a sulfur source [27].…”