title: Role of the [2Fe-2S]2+ cluster in biotin synthase: mutagenesis of the atypical metal ligand arginine 260.
abstract: Biotin synthase (BS) is an S-adenosylmethionine (AdoMet)-dependent radical enzyme that catalyzes the addition of sulfur to dethiobiotin. Like other AdoMet radical enzymes, BS contains a [4Fe-4S] cluster that is coordinated by a highly conserved CxxxCxxC sequence motif and by the methionyl amine and carboxylate of AdoMet. The close association of the [4Fe-4S]+ cluster with AdoMet facilitates reductive cleavage of the sulfonium and the generation of transient 5'-deoxyadenosyl radicals, which are then proposed to sequentially abstract hydrogen atoms from the substrate to produce carbon radicals at C9 and C6 of dethiobiotin. BS also contains a [2Fe-2S]2+ cluster located approximately 4-5 A from dethiobiotin, and we have proposed that a bridging sulfide of this cluster quenches the substrate radicals, leading to formation of the thiophane ring of biotin. In BS from Escherichia coli, the [2Fe-2S]2+ cluster is coordinated by cysteines 97, 128, and 188, and the atypical metal ligand, arginine 260. The evolutionary conservation of an arginine guanidinium as a metal ligand suggests a novel role for this residue in tuning the reactivity or stability of the [2Fe-2S]2+ cluster. In this work, we explore the effects of mutagenesis of Arg260 to Ala, Cys, His, and Met. Although perturbations in a number of characteristics of the [2Fe-2S]2+ cluster and the proteins are noted, the reconstituted enzymes have in vitro single-turnover activities that are 30-120% of that of the wild type. Further, in vivo expression of each mutant enzyme was sufficient to sustain growth of a bioB- mutant strain on dethiobiotin-supplemented medium, suggesting the enzymes were active and efficiently reconstituted by the in vivo iron-sulfur cluster (ISC) assembly system. Although we cannot exclude an as-yet-unidentified in vivo role in cluster repair or retention, we can conclude that Arg260 is not essential for the catalytic reaction of BS.
authors.affiliation: Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Robyn B
authors.initials: RB
authors.lastname: Broach
authors.forename: Joseph T
authors.initials: JT
authors.lastname: Jarrett
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D008024:Ligands; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017386:Sequence Homology, Amino Acid; D013466:Sulfurtransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D008024:Ligands; D001120:Arginine; D013466:Sulfurtransferases; C057985:biotin synthetase