. BioB The b e noire of biotin synthesis has long been

. BioB The b e noire of biotin synthesis has long been the last step, insertion of the sulfur atom into DTB to form the thiophane ring of biotin. For many years this activity was ascribed to BioB by genetic means (biotin auxotrophs unable to grow on DTB) and could be assayed only by the ability of intact cells to convert DTB to biotin. Extensive attempts to obtain sulfur insertion in vitro all failed until Ifuku and coworkers (51) succeeded in showing biotin synthesis from DTB in a cell extract. The reaction required DTB, SAM, NADPH, BioB, and an unknown protein or proteins later shown to be flavodoxin (FldA) and flavodoxin reductase (Fpr) (51?3). This breakthrough was soon followed by demonstration of activity in a defined system containing NADPH, flavodoxin and flavodoxin reductase as the electron transfer system plus DTB, SAM, and a BioB preparation plus a reducing environment (54). BioB (a homodimer of a 38.6 kDa subunit) was found to be a very labile protein that is best purified and assayed under anaerobic conditions. The discovery that SAM was absolutely required for biotin synthesis and was not the sulfur donor (55, 56) strongly suggested that BioB was a member of the (then) small family of “radical SAM” enzymes. It has recently become apparent that this is a large family of proteins that catalyze a range of reactions that invariably involve difficult reactions often accessible only by radical chemistry. The radical is generated by reductive cleavage of SAM to give a deoxyadenosyl radical (DOA? plus methionine. The DOA radical then PNPP web cleaves a C–H bond to generate a carbon radical that allows the chemistry to proceed. The electron donor in the single electron reduction of SAM is a [4Fe-4S] cluster liganded to the cysteine residues of a perfectly conserved CXXXCXXC motif. Consistent with this picture, the BioB reaction is chemically difficult since it requires cleavage and sulfur insertion into two unreactive C–H bonds. The mechanism currently accepted by most workers in the field (57?9) is given in Fig. 4. The BioB species involved in the mechanism of Fig. 4 contains two [Fe-S] clusters. The number and composition of these clusters has been the subject of much disagreement in the literature. However, a variety of spectroscopic techniques plus a recent BioB crystal structure give a consistent picture. BioB contains two different clusters, the [4Fe-4S] cluster characteristic of radical SAM enzymes and a [order Crotaline 2Fe-2S] cluster located at a different site (the [2Fe-2S] cluster was often thought to be a degradation product of a [4Fe-4S] cluster until mutagenesis experiments suggested otherwise). The crystal structure shows that BioB to be a /8 (TIM) barrel protein with the two [Fe-S] clusters located at either end of the barrel (58). The [4Fe-4S] cluster is located at the open end of the barrel whereas the [2Fe-2S] cluster (which utilizes an unusual arginine ligand) is at the closed end of the barrel. TheAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptEcoSal Plus. Author manuscript; available in PMC 2015 January 06.CronanPagecrystal structures contain both SAM and DTB. 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, 9mercaptodethiobiotin is a catalytic intermediate (60?2) This latter finding fits with the belief of most workers in the field that the [2Fe-.. BioB The b e noire of biotin synthesis has long been the last step, insertion of the sulfur atom into DTB to form the thiophane ring of biotin. For many years this activity was ascribed to BioB by genetic means (biotin auxotrophs unable to grow on DTB) and could be assayed only by the ability of intact cells to convert DTB to biotin. Extensive attempts to obtain sulfur insertion in vitro all failed until Ifuku and coworkers (51) succeeded in showing biotin synthesis from DTB in a cell extract. The reaction required DTB, SAM, NADPH, BioB, and an unknown protein or proteins later shown to be flavodoxin (FldA) and flavodoxin reductase (Fpr) (51?3). This breakthrough was soon followed by demonstration of activity in a defined system containing NADPH, flavodoxin and flavodoxin reductase as the electron transfer system plus DTB, SAM, and a BioB preparation plus a reducing environment (54). BioB (a homodimer of a 38.6 kDa subunit) was found to be a very labile protein that is best purified and assayed under anaerobic conditions. The discovery that SAM was absolutely required for biotin synthesis and was not the sulfur donor (55, 56) strongly suggested that BioB was a member of the (then) small family of “radical SAM” enzymes. It has recently become apparent that this is a large family of proteins that catalyze a range of reactions that invariably involve difficult reactions often accessible only by radical chemistry. The radical is generated by reductive cleavage of SAM to give a deoxyadenosyl radical (DOA? plus methionine. The DOA radical then cleaves a C–H bond to generate a carbon radical that allows the chemistry to proceed. The electron donor in the single electron reduction of SAM is a [4Fe-4S] cluster liganded to the cysteine residues of a perfectly conserved CXXXCXXC motif. Consistent with this picture, the BioB reaction is chemically difficult since it requires cleavage and sulfur insertion into two unreactive C–H bonds. The mechanism currently accepted by most workers in the field (57?9) is given in Fig. 4. The BioB species involved in the mechanism of Fig. 4 contains two [Fe-S] clusters. The number and composition of these clusters has been the subject of much disagreement in the literature. However, a variety of spectroscopic techniques plus a recent BioB crystal structure give a consistent picture. BioB contains two different clusters, the [4Fe-4S] cluster characteristic of radical SAM enzymes and a [2Fe-2S] cluster located at a different site (the [2Fe-2S] cluster was often thought to be a degradation product of a [4Fe-4S] cluster until mutagenesis experiments suggested otherwise). The crystal structure shows that BioB to be a /8 (TIM) barrel protein with the two [Fe-S] clusters located at either end of the barrel (58). The [4Fe-4S] cluster is located at the open end of the barrel whereas the [2Fe-2S] cluster (which utilizes an unusual arginine ligand) is at the closed end of the barrel. TheAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptEcoSal Plus. Author manuscript; available in PMC 2015 January 06.CronanPagecrystal structures contain both SAM and DTB. 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, 9mercaptodethiobiotin is a catalytic intermediate (60?2) This latter finding fits with the belief of most workers in the field that the [2Fe-.