Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
Cell Chem Biol. 2016 Sep 22;23(9):1091-1097. doi: 10.1016/j.chembiol.2016.07.018. Epub 2016 Sep 8.
SimC7 is a polyketide ketoreductase involved in biosynthesis of the angucyclinone moiety of the gyrase inhibitor simocyclinone D8 (SD8). SimC7, which belongs to the short-chain dehydrogenase/reductase (SDR) superfamily, catalyzes reduction of the C-7 carbonyl of the angucyclinone, and the resulting hydroxyl is essential for antibiotic activity. SimC7 shares little sequence similarity with characterized ketoreductases, suggesting it might have a distinct mechanism. To investigate this possibility, we determined the structures of SimC7 alone, with NADP(+), and with NADP(+) and the substrate 7-oxo-SD8. These structures show that SimC7 is distinct from previously characterized polyketide ketoreductases, lacking the conserved catalytic triad, including the active-site tyrosine that acts as central acid-base catalyst in canonical SDR proteins. Taken together with functional analyses of active-site mutants, our data suggest that SimC7 catalyzes a substrate-assisted, two-step reaction for reduction of the C-7 carbonyl group involving intramolecular transfer of a substrate-derived proton to generate a phenolate intermediate.
SimC7 是一种聚酮酮还原酶，参与了柔红霉素抑制剂 simocyclinone D8（SD8）中环庚酮部分的生物合成。SimC7 属于短链脱氢酶/还原酶（SDR）超家族，催化环庚酮 C-7 羰基的还原，生成的羟基对于抗生素活性至关重要。SimC7 与已鉴定的酮还原酶序列相似性较低，表明它可能具有独特的机制。为了研究这种可能性，我们分别测定了 SimC7 单体、与 NADP(+)、以及与 NADP(+)和底物 7-氧代-SD8 结合的结构。这些结构表明，SimC7 与先前鉴定的聚酮酮还原酶不同，缺乏保守的催化三联体，包括在典型的 SDR 蛋白中作为中心酸碱催化剂的活性位点酪氨酸。与活性位点突变体的功能分析相结合，我们的数据表明，SimC7 催化一个涉及底物辅助的两步反应，通过将底物衍生的质子进行分子内转移来还原 C-7 羰基，从而生成酚盐中间体。
