Allen J R, Ensign S A
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300, USA.
Biochemistry. 1999 Jan 5;38(1):247-56. doi: 10.1021/bi982114h.
Epoxide carboxylase from the bacterium Xanthobacter strain Py2 is a multicomponent enzyme system which catalyzes the pyridine nucleotide-dependent carboxylation of aliphatic epoxides to beta-ketoacids as illustrated by the reaction epoxypropane + CO2 + NADPH + NAD+ --> acetoacetate + H+ + NADP+ + NADH. The combination of four distinct proteins, designated components I-IV, are required for the reconstitution of epoxide carboxylase activity with racemic mixtures of short-chain (C3-C5) terminal epoxyalkanes. In this work, components III and IV of the epoxide carboxylase system are shown to confer specificity for epoxyalkane enantiomers. Components I-III supported the carboxylation of (R)-epoxypropane, while components I, II, and IV supported the carboxylation of (S)-epoxypropane. At fixed concentrations of components I and II, the rates of (R)- and (S)-epoxypropane carboxylation saturated with increasing concentrations of component III or IV to give identical maximal rates for the two epoxide substrates. (S)-Epoxypropane was an inactivator of (R)-epoxypropane carboxylation by components I- III, while (R)-epoxypropane was an inactivator of (S)-epoxypropane carboxylation by components I, II, and IV. These inactivating effects were fully reversed upon the addition of the correct complementing dehydrogenase component. Amino acid sequence analysis of components III and IV demonstrates that they belong to the short-chain dehydrogenase/reductase (SDR) family of enzymes. Both components contain highly conserved residues within the coenzyme binding fold and catalytic regions found in SDR enzymes. Components III and IV are proposed to catalyze the NAD+-dependent abstraction of a hydride from a chiral secondary alcohol-like intermediate bound to the active site component of the enzyme system to form the corresponding beta-ketone intermediate. A multicomponent epoxide carboxylase system was purified to homogeneity from Nocardia corallina B276, a bacterium phylogenetically unrelated to Xanthobacter Py2, and found to consist of four proteins with functions identical to those of the Xanthobacter Py2 system. The stereoselective dehydrogenases of the Xanthobacter epoxide carboxylase system were able to substitute for the corresponding components of the N. corallina system when using (R)- and (S)-epoxypropane as substrates, and vice versa. These results provide the first demonstration of the involvement of stereospecific dehydrogenases in aliphatic epoxide metabolism and provide new insights into microbial strategies for the utilization of chiral organic molecules.
来自黄杆菌属菌株Py2的环氧化物羧化酶是一种多组分酶系统，它催化脂肪族环氧化物在吡啶核苷酸依赖下羧化为β-酮酸，反应式如下：环氧丙烷 + CO₂ + NADPH + NAD⁺ → 乙酰乙酸 + H⁺ + NADP⁺ + NADH。短链（C3 - C5）末端环氧烷烃的外消旋混合物重建环氧化物羧化酶活性需要四种不同的蛋白质（称为组分I - IV）组合。在这项工作中，环氧化物羧化酶系统的组分III和IV显示出对环氧烷对映体具有特异性。组分I - III支持（R）-环氧丙烷的羧化反应，而组分I、II和IV支持（S）-环氧丙烷的羧化反应。在组分I和II浓度固定时，随着组分III或IV浓度增加，（R）-和（S）-环氧丙烷羧化反应速率达到饱和，两种环氧化物底物的最大反应速率相同。（S）-环氧丙烷是组分I - III催化（R）-环氧丙烷羧化反应的抑制剂，而（R）-环氧丙烷是组分I、II和IV催化（S）-环氧丙烷羧化反应的抑制剂。加入正确的互补脱氢酶组分后，这些抑制作用完全逆转。组分III和IV的氨基酸序列分析表明它们属于短链脱氢酶/还原酶（SDR）家族。两种组分在辅酶结合折叠区和SDR酶的催化区域内都含有高度保守的残基。推测组分III和IV催化从与酶系统活性位点组分结合的手性仲醇样中间体上以NAD⁺依赖方式提取一个氢负离子，形成相应的β-酮中间体。从珊瑚诺卡氏菌B276（一种与黄杆菌Py2在系统发育上无关的细菌）中纯化得到了一个多组分环氧化物羧化酶系统，该系统由四种功能与黄杆菌Py2系统相同的蛋白质组成。当以（R）-和（S）-环氧丙烷为底物时，黄杆菌环氧化物羧化酶系统的立体选择性脱氢酶能够替代珊瑚诺卡氏菌系统的相应组分，反之亦然。这些结果首次证明了立体特异性脱氢酶参与脂肪族环氧化物代谢，并为微生物利用手性有机分子的策略提供了新见解。
