Kurteva-Yaneva Nadya, Zahn Michael, Weichler M-Teresa, Starke Robert, Harms Hauke, Müller Roland H, Sträter Norbert, Rohwerder Thore
From the Department of Environmental Microbiology, Helmholtz Centre for Environmental Research (UFZ), 04318 Leipzig and.
the Center for Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, University of Leipzig, 04103 Leipzig, Germany.
J Biol Chem. 2015 Apr 10;290(15):9727-37. doi: 10.1074/jbc.M115.645689. Epub 2015 Feb 26.
Bacterial coenzyme B12-dependent 2-hydroxyisobutyryl-CoA mutase (HCM) is a radical enzyme catalyzing the stereospecific interconversion of (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA. It consists of two subunits, HcmA and HcmB. To characterize the determinants of substrate specificity, we have analyzed the crystal structure of HCM from Aquincola tertiaricarbonis in complex with coenzyme B12 and the substrates (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in alternative binding. When compared with the well studied structure of bacterial and mitochondrial B12-dependent methylmalonyl-CoA mutase (MCM), HCM has a highly conserved domain architecture. However, inspection of the substrate binding site identified amino acid residues not present in MCM, namely HcmA Ile(A90) and Asp(A117). Asp(A117) determines the orientation of the hydroxyl group of the acyl-CoA esters by H-bond formation, thus determining stereospecificity of catalysis. Accordingly, HcmA D117A and D117V mutations resulted in significantly increased activity toward (R)-3-hydroxybutyryl-CoA. Besides interconversion of hydroxylated acyl-CoA esters, wild-type HCM as well as HcmA I90V and I90A mutant enzymes could also isomerize pivalyl- and isovaleryl-CoA, albeit at >10 times lower rates than the favorite substrate (S)-3-hydroxybutyryl-CoA. The nonconservative mutation HcmA D117V, however, resulted in an enzyme showing high activity toward pivalyl-CoA. Structural requirements for binding and isomerization of highly branched acyl-CoA substrates such as 2-hydroxyisobutyryl- and pivalyl-CoA, possessing tertiary and quaternary carbon atoms, respectively, are discussed.
细菌辅酶B12依赖性2-羟基异丁酰辅酶A变位酶（HCM）是一种自由基酶，催化（S）-3-羟基丁酰辅酶A和2-羟基异丁酰辅酶A的立体特异性相互转化。它由两个亚基HcmA和HcmB组成。为了表征底物特异性的决定因素，我们分析了来自第三碳源嗜水菌的HCM与辅酶B12以及交替结合的底物（S）-3-羟基丁酰辅酶A和2-羟基异丁酰辅酶A形成的复合物的晶体结构。与已深入研究的细菌和线粒体B12依赖性甲基丙二酰辅酶A变位酶（MCM）的结构相比，HCM具有高度保守的结构域结构。然而，对底物结合位点的检查发现了MCM中不存在的氨基酸残基，即HcmA Ile（A90）和Asp（A117）。Asp（A117）通过氢键形成决定了酰基辅酶A酯羟基的取向，从而决定了催化的立体特异性。因此，HcmA D117A和D117V突变导致对（R）-3-羟基丁酰辅酶A的活性显著增加。除了羟基化酰基辅酶A酯的相互转化外，野生型HCM以及HcmA I90V和I90A突变酶也能使新戊酰辅酶A和异戊酰辅酶A异构化，尽管其速率比最适底物（S）-3-羟基丁酰辅酶A低10倍以上。然而，非保守突变HcmA D117V导致一种对新戊酰辅酶A具有高活性的酶。本文讨论了分别具有叔碳原子和季碳原子的高度支链酰基辅酶A底物如2-羟基异丁酰辅酶A和新戊酰辅酶A结合和异构化的结构要求。
