Jost Marco, Born David A, Cracan Valentin, Banerjee Ruma, Drennan Catherine L
Departments of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
Departments of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; the Graduate Program in Biophysics, Harvard University, Cambridge, Massachusetts 02138.
J Biol Chem. 2015 Nov 6;290(45):26882-26898. doi: 10.1074/jbc.M115.676890. Epub 2015 Aug 28.
Acyl-CoA mutases are a growing class of adenosylcobalamin-dependent radical enzymes that perform challenging carbon skeleton rearrangements in primary and secondary metabolism. Members of this class of enzymes must precisely control substrate positioning to prevent oxidative interception of radical intermediates during catalysis. Our understanding of substrate specificity and catalysis in acyl-CoA mutases, however, is incomplete. Here, we present crystal structures of IcmF, a natural fusion protein variant of isobutyryl-CoA mutase, in complex with the adenosylcobalamin cofactor and four different acyl-CoA substrates. These structures demonstrate how the active site is designed to accommodate the aliphatic acyl chains of each substrate. The structures suggest that a conformational change of the 5'-deoxyadenosyl group from C2'-endo to C3'-endo could contribute to initiation of catalysis. Furthermore, detailed bioinformatic analyses guided by our structural findings identify critical determinants of acyl-CoA mutase substrate specificity and predict new acyl-CoA mutase-catalyzed reactions. These results expand our understanding of the substrate specificity and the catalytic scope of acyl-CoA mutases and could benefit engineering efforts for biotechnological applications ranging from production of biofuels and commercial products to hydrocarbon remediation.
酰基辅酶A变位酶是一类不断增加的依赖腺苷钴胺素的自由基酶，它们在初级和次级代谢中进行具有挑战性的碳骨架重排。这类酶的成员必须精确控制底物定位，以防止催化过程中自由基中间体的氧化拦截。然而，我们对酰基辅酶A变位酶的底物特异性和催化作用的理解并不完整。在此，我们展示了异丁酰辅酶A变位酶的天然融合蛋白变体IcmF与腺苷钴胺素辅因子和四种不同酰基辅酶A底物形成复合物的晶体结构。这些结构展示了活性位点是如何设计以容纳每种底物的脂肪族酰基链的。这些结构表明，5'-脱氧腺苷基团从C2'-内型到C3'-内型的构象变化可能有助于催化作用的启动。此外，在我们的结构发现指导下进行的详细生物信息学分析确定了酰基辅酶A变位酶底物特异性的关键决定因素，并预测了新的酰基辅酶A变位酶催化的反应。这些结果扩展了我们对酰基辅酶A变位酶的底物特异性和催化范围的理解，并可能有益于从生物燃料和商业产品生产到碳氢化合物修复等生物技术应用的工程努力。
