Imer produced 35 biotin over 4 h indicatingAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptEcoSal Plus. Author manuscript; available in PMC 2015 January 06.CronanPageat least three consecutive turnovers (74). Biotin production showed burst kinetics, a burst phase of k = 0.12 min-1, followed by a steady-state phase with a turnover number of k = 0.0089 min-1. The rate of the burst phase observed in vitro is similar to that observed in vivo suggesting that in vivo activity is not limited by FeS cluster reassembly but rather by the chemistry of biotin formation. The key to obtaining catalysis in vitro was preparation of SAM free of the contaminents present in commercial preparations and addition of Mtn to cleave the inhibitory 5-deoxyadenosine produced in the reaction. It should be noted that the in vivo measurement of BioB catalysis was complicated by the unexpected finding that enzyme turnover renders the enzyme susceptible to proteolytic degradation (73). A 50 ?0 depletion of the level of His6-BioB was observed after incubation. This depletion was not observed in the absence of DTB or in the presence of biotin (73). The observed degradation of BioB was proposed to result from collapse of the enzyme [2Fe-2S] center due to donation of a sulfur atom to DTB. The [2Fe-2S] centre of BioB is located deep within the barrel of this /8 (TIM) protein (58) and thus it seems probable that a substantial unfolding of the protein would be required to allow rebuilding of the [2Fe-2S] cluster. Such unfolding would allow restoration of the [2Fe-2S] center, but at the cost of exposure of the protein to proteolytic attack while unfolded. Therefore, in this scenario catalysis by a molecule of BioB would require the protein to run a gauntlet of proteolysis until restoration of normal folding (with concomitant resistance to proteolysis) by rebuilding of the [2Fe-2S] center expended in biotin synthesis (73). The turnover numbers observed may thus be viewed as the products of a stochastic process. If the [2Fe-2S] cluster of a BioB molecule is rebuilt before proteolysis occurs, that protein will perform another turnover. If not, the protein molecule perishes and must be re-synthesized de novo. More recent work done in vitro has shown that loss of iron-sulfur clusters from BioB as a result of catalysis promotes unfolding and degradation (75). Hence, some BioB molecules may catalyze only one or a few turnovers in their lifetimes whereas others may complete >100 turnovers. The steady state level following the burst phase in the optimized in vitro system (74) may reflect the loss of active BioB molecules. The biotin requirement of mtn (pfs) mutant strains (20) is due to inhibition of BioB by the GS-5816 mechanism of action byproduct of sulfur insertion, 5-deoxyadenosine (21). The mtn gene encodes the 5methylthioadenosine/S-adenosylhomocysteine nucleosidase which was shown to also cleave 5-deoxyadenosine to adenine plus 5-deoxyribose (21, 74, 76). Mutants lacking Mtn activity precisely mimic BioB mutants in that they grow on biotin, but not on DTB or DAPA, and buy Cyclopamine excrete DTB (21).Author Manuscript Author Manuscript Author Manuscript Author ManuscriptRemaining problems in biotin synthesisThe E. coli bioH gene differs from the other genes in the pathway in that it is neither located within the bio operon nor regulated by the BirA repressor/biotin protein ligase (77, 78). This is in contrast to many other bacteria where bioH resides within the biotin operon and is generally lo.Imer produced 35 biotin over 4 h indicatingAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptEcoSal Plus. Author manuscript; available in PMC 2015 January 06.CronanPageat least three consecutive turnovers (74). Biotin production showed burst kinetics, a burst phase of k = 0.12 min-1, followed by a steady-state phase with a turnover number of k = 0.0089 min-1. The rate of the burst phase observed in vitro is similar to that observed in vivo suggesting that in vivo activity is not limited by FeS cluster reassembly but rather by the chemistry of biotin formation. The key to obtaining catalysis in vitro was preparation of SAM free of the contaminents present in commercial preparations and addition of Mtn to cleave the inhibitory 5-deoxyadenosine produced in the reaction. It should be noted that the in vivo measurement of BioB catalysis was complicated by the unexpected finding that enzyme turnover renders the enzyme susceptible to proteolytic degradation (73). A 50 ?0 depletion of the level of His6-BioB was observed after incubation. This depletion was not observed in the absence of DTB or in the presence of biotin (73). The observed degradation of BioB was proposed to result from collapse of the enzyme [2Fe-2S] center due to donation of a sulfur atom to DTB. The [2Fe-2S] centre of BioB is located deep within the barrel of this /8 (TIM) protein (58) and thus it seems probable that a substantial unfolding of the protein would be required to allow rebuilding of the [2Fe-2S] cluster. Such unfolding would allow restoration of the [2Fe-2S] center, but at the cost of exposure of the protein to proteolytic attack while unfolded. Therefore, in this scenario catalysis by a molecule of BioB would require the protein to run a gauntlet of proteolysis until restoration of normal folding (with concomitant resistance to proteolysis) by rebuilding of the [2Fe-2S] center expended in biotin synthesis (73). The turnover numbers observed may thus be viewed as the products of a stochastic process. If the [2Fe-2S] cluster of a BioB molecule is rebuilt before proteolysis occurs, that protein will perform another turnover. If not, the protein molecule perishes and must be re-synthesized de novo. More recent work done in vitro has shown that loss of iron-sulfur clusters from BioB as a result of catalysis promotes unfolding and degradation (75). Hence, some BioB molecules may catalyze only one or a few turnovers in their lifetimes whereas others may complete >100 turnovers. The steady state level following the burst phase in the optimized in vitro system (74) may reflect the loss of active BioB molecules. The biotin requirement of mtn (pfs) mutant strains (20) is due to inhibition of BioB by the byproduct of sulfur insertion, 5-deoxyadenosine (21). The mtn gene encodes the 5methylthioadenosine/S-adenosylhomocysteine nucleosidase which was shown to also cleave 5-deoxyadenosine to adenine plus 5-deoxyribose (21, 74, 76). Mutants lacking Mtn activity precisely mimic BioB mutants in that they grow on biotin, but not on DTB or DAPA, and excrete DTB (21).Author Manuscript Author Manuscript Author Manuscript Author ManuscriptRemaining problems in biotin synthesisThe E. coli bioH gene differs from the other genes in the pathway in that it is neither located within the bio operon nor regulated by the BirA repressor/biotin protein ligase (77, 78). This is in contrast to many other bacteria where bioH resides within the biotin operon and is generally lo.
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