Biotin is an essential vitamin used as a cofactor in carboxylation reactions central to human metabolism, particularly in enzymes involved in fatty acid biosynthesis, gluconeogenesis, and branched-chain amino acid catabolism. Biotin is biosynthesized in microbes and plants through a four-step pathway that terminates with addition of sulfur and formation of the thiophane ring. This sulfur insertion is an impressive feat of enzyme catalysis that requires removal of two unactivated hydrogen atoms from the substrate dethiobiotin. In E. coli, sulfur insertion is catalyzed by the homodimeric iron-sulfur protein biotin synthase (BS), requires flavodoxin and S-adenosylmethionine (AdoMet), and produces methionine and 5'-deoxyadenosine. Collectively these traits indicate that biotin synthase is an AdoMet-dependent radical enzyme that catalyzes reductive cleavage of the AdoMet sulfonium to produce 5'-deoxyadenosyl radicals. We have proposed and provided evidence for a mechanism in which AdoMet coordinates a [4Fe-4S] 2+ cluster and subsequent electron transfer into the AdoMet/[4Fe-4S] 2+ cluster complex results in production of a 5'-deoxyadenosyl radical. This radical abstracts a hydrogen atom from the substrate, dethiobiotin, generating a substrate carbon radical. The substrate radical is quenched by a sulfur atom from the [2Fe-2S] 2+ cluster, generating 9-mercaptodethiobiotin as a discrete intermediate. A second AdoMet cleavage triggers a similar reaction sequence, leading to formation of the second C-S bond in the product biotin. We have solved the crystal structure of E. coil biotin synthase with substrates bound and the relative positions of the respective reactants strongly supports this mechanistic proposal. Armed with knowledge derived from the structure, along with the expertise we have developed in anaerobic biochemistry, we are poised to test several key aspects of this mechanism. First, we will use the structure as a guide and test the roles of several conserved protein residues in catalysis. Second, we have developed a half-turnover reaction that will allow us to study the formation of 9-mercaptodethiobiotin and the associated iron-sulfur cluster states using stopped-flow and quench-flow kinetic methods. Finally, since the mechanism we propose describes a single turnover that results in destruction of the [2Fe-2S] 2+ cluster, we are examining possible mechanisms for cluster reassembly that facilitate multiple enzyme turnovers in vivo. A detailed knowledge of the chemical requirements for production of 5'-deoxyadenosyl radicals and for controlled utilization of these radicals for substrate activation and biotin formation will contribute to our expanding understanding of the general features common to all radical enzymes.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Physical Biochemistry Study Section (PB)
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Jones, Warren
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University of Hawaii
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Reyda, Michael R; Fugate, Corey J; Jarrett, Joseph T (2009) A complex between biotin synthase and the iron-sulfur cluster assembly chaperone HscA that enhances in vivo cluster assembly. Biochemistry 48:10782-92
Farrar, Christine E; Jarrett, Joseph T (2009) Protein residues that control the reaction trajectory in S-adenosylmethionine radical enzymes: mutagenesis of asparagine 153 and aspartate 155 in Escherichia coli biotin synthase. Biochemistry 48:2448-58
Reyda, Michael R; Dippold, Rachael; Dotson, Michael E et al. (2008) Loss of iron-sulfur clusters from biotin synthase as a result of catalysis promotes unfolding and degradation. Arch Biochem Biophys 471:32-41
Taylor, Andrew M; Farrar, Christine E; Jarrett, Joseph T (2008) 9-Mercaptodethiobiotin is formed as a competent catalytic intermediate by Escherichia coli biotin synthase. Biochemistry 47:9309-17
Broach, Robyn B; Jarrett, Joseph T (2006) Role of the [2Fe-2S]2+ cluster in biotin synthase: mutagenesis of the atypical metal ligand arginine 260. Biochemistry 45:14166-74
Jarrett, Joseph T (2005) The novel structure and chemistry of iron-sulfur clusters in the adenosylmethionine-dependent radical enzyme biotin synthase. Arch Biochem Biophys 433:312-21
Jarrett, Joseph T (2005) Biotin synthase: enzyme or reactant? Chem Biol 12:409-10
Berkovitch, Frederick; Nicolet, Yvain; Wan, Jason T et al. (2004) Crystal structure of biotin synthase, an S-adenosylmethionine-dependent radical enzyme. Science 303:76-9
Jarrett, Joseph T (2003) The generation of 5'-deoxyadenosyl radicals by adenosylmethionine-dependent radical enzymes. Curr Opin Chem Biol 7:174-82
Ugulava, Natalia B; Frederick, Kendra K; Jarrett, Joseph T (2003) Control of adenosylmethionine-dependent radical generation in biotin synthase: a kinetic and thermodynamic analysis of substrate binding to active and inactive forms of BioB. Biochemistry 42:2708-19

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