Bruender Nathan A, Young Anthony P, Bandarian Vahe
Department of Chemistry and Biochemistry, The University of Arizona, Tucson, Arizona 85721-0088, United States.
Biochemistry. 2015 May 12;54(18):2903-10. doi: 10.1021/acs.biochem.5b00210. Epub 2015 May 1.
The radical S-adenosyl-L-methionine (SAM) superfamily is a large and growing group of enzymes that conduct complex radical-mediated transformations. A one-electron reduction of SAM via the +1 state of the cubane [4Fe-4S] cluster generates a 5'-deoxyadenosyl radical, which initiates turnover. The [4Fe-4S] cluster must be reduced from its resting +2 state to the catalytically active +1 oxidation state by an electron. In practice, dithionite or the Escherichia coli flavodoxin (EcFldA)/ferredoxin (flavodoxin):NADP(+) oxidoreductase (Fpr)/NADPH system is used. Herein, we present a systematic investigation of the reductive activation of the radical SAM enzyme CDG synthase (BsQueE) from Bacillus subtilis comparing biological and chemical reductants. These data show that either of the flavodoxin homologues encoded by the B. subtilis genome, BsYkuN or BsYkuP, as well as a series of small molecule redox mediators, supports BsQueE activity. With dithionite as a reductant, the activity of BsQueE is ~75-fold greater in the presence of BsYkuN and BsYkuP compared to that in the presence of dithionite alone. By contrast, EcFldA supports turnover to ~10-fold greater levels than dithionite alone under the same conditions. Comparing the ratio of the rate of turnover to the apparent binding constant for the flavodoxin homologues reveals 10- and 240-fold preferences for BsYkuN over BsYkuP and EcFldA, respectively. The differential activation of the enzyme cannot be explained by the abortive cleavage of SAM. We conclude from these observations that the differential activation of BsQueE by Fld homologues may reside in the details of the interaction between the flavodoxin and the radical SAM enzyme.
自由基S-腺苷-L-甲硫氨酸（SAM）超家族是一类庞大且不断增加的酶，它们能进行复杂的自由基介导的转化反应。通过立方烷型[4Fe-4S]簇的+1价态对SAM进行单电子还原会产生一个5'-脱氧腺苷自由基，从而启动催化循环。[4Fe-4S]簇必须通过一个电子从其静止的+2价态还原为催化活性的+1氧化态。实际上，常用连二亚硫酸盐或大肠杆菌黄素氧还蛋白（EcFldA）/铁氧还蛋白（黄素氧还蛋白）：NADP(+)氧化还原酶（Fpr）/NADPH系统。在此，我们对枯草芽孢杆菌的自由基SAM酶CDG合酶（BsQueE）的还原激活进行了系统研究，比较了生物还原剂和化学还原剂。这些数据表明，枯草芽孢杆菌基因组编码的黄素氧还蛋白同系物BsYkuN或BsYkuP，以及一系列小分子氧化还原介质，都能支持BsQueE的活性。以连二亚硫酸盐作为还原剂时，与单独使用连二亚硫酸盐相比，在存在BsYkuN和BsYkuP的情况下，BsQueE的活性高出约75倍。相比之下，在相同条件下，EcFldA支持的催化循环水平比单独使用连二亚硫酸盐时高出约10倍。比较黄素氧还蛋白同系物的催化循环速率与表观结合常数的比值可知，BsYkuN对BsYkuP和EcFldA的偏好分别为10倍和240倍。酶的差异激活不能用SAM的无效裂解来解释。我们从这些观察结果得出结论，黄素氧还蛋白同系物对BsQueE的差异激活可能存在于黄素氧还蛋白与自由基SAM酶之间相互作用的细节中。
