Abstract

Biotin is an essential protein cofactor used in carboxylation reactions central to human metabolism including enzymes involved in fatty acid biosynthesis, gluconeogenesis, and branched-chain amino acid catabolism. The terminal step in biotin biosynthesis involves the insertion of a sulfur atom between C6 and C9 of the precursor dethiobiotin, forming the biotin thioether ring. This insertion reaction is deceptively simple yet represents an impressive feat of enzymatic catalysis, requiring the enzyme break two saturated, inactivated CH bonds in dethiobiotin prior to sulfur insertion. This reaction is catalyzed by the E. Coli BioB protein, a dimeric iron- sulfur protein, and requires the participation of adenosylmethionine (AdoMet) and reduced flavodoxin. The requirement for AdoMet and flavodoxin suggests that biotin synthase is a member of a family of enzymes that reductively cleave AdoMet to generate a 5' - deoxyadenosyl radical, which is then used to generate a protein radical or to directly abstract a hydrogen atom from the substrate. More generally, biotin synthase belongs to a class of enzymes that are able to generate and control carbon and/or sulfur radicals in order to carry out difficult biosynthetic transformations; these enzymes include the human enzymes ribonucleotide reductase and lipoic acid synthase. Several obstacles have hindered in depth studies of the mechanism of biotin thioether ring formation: (I) the sulfur- containing substrate for BioB is not known, (II) the redox requirements for thioether ring formation are not known, and (III) the role of AdoMet and flavodoxin in enzyme activation is not understood. Studies described in this proposal address these substrate, protein, and activation requirements for biotin thioether ring formation, and will lead to more detailed studies of the chemical mechanism of radical-mediated sulfur insertion. By integrating information from these studies our knowledge of other enzymes in the biotin biosynthetic pathway, reconstitution of essential components of this pathway may in the future provide a convenient and inexpensive source of biotin for nutritional, therapeutic, and industrial applications.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059175-03
Application #
6386438
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Preusch, Peter C
Project Start
1999-04-01
Project End
2004-03-31
Budget Start
2001-04-01
Budget End
2002-03-31
Support Year
3
Fiscal Year
2001
Total Cost
$218,992
Indirect Cost

  Institution

Name
University of Pennsylvania
Department
Biochemistry
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104

  Publications

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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