Small F J, Ensign S A
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300, USA.
J Biol Chem. 1997 Oct 3;272(40):24913-20. doi: 10.1074/jbc.272.40.24913.
Alkene monooxygenase from Xanthobacter strain Py2 is an inducible enzyme that catalyzes the O2- and NADH-dependent epoxidation of short chain (C2 to C6) alkenes to their corresponding epoxides as the initial step in the utilization of aliphatic alkenes as carbon and energy sources. In the present study, alkene monooxygenase has been resolved from the soluble fraction of cell-free extracts into four components, each of which has been purified to homogeneity, that are obligately required for alkene epoxidation activity. The four required components are 1) a monomeric 35.5-kDa protein containing 1 mol of FAD and a probable 2Fe-2S center; 2) a 13.3-kDa ferredoxin containing a Rieske-type 2Fe-2S cluster; 3) an 11-kDa monomeric protein that contains no detectable cofactors; and 4) a 212-kDa alpha2beta2gamma2 multimeric protein containing four atoms of nonheme iron. The 35.5-kDa protein has been characterized as an NADH reductase. The physiological electron acceptor for the reductase was the Rieske-type ferredoxin, which is proposed to be an intermediate electron carrier between the reductase and terminal catalytic component of the system. The 212-kDa protein was specifically inactivated in cell-free extracts by the mechanism-based inactivator propyne, suggesting that it is the catalytic component and contains the active site(s) for O2 activation and alkene epoxidation. The subunit structure and metal analysis of this component suggest that it contains two diiron centers, one for each alphabetagamma protomeric unit. No specific enzymatic activities could be assigned for the 11-kDa protein, but this component was obligately required for steady-state alkene epoxidation. The alkene monooxygenase components were expressed during growth of Xanthobacter Py2 on aliphatic alkenes or epoxides and repressed during growth on other carbon sources. The electron transfer components of alkene monooxygenase were highly specific: other reductase activities present in Xanthobacter were incapable of transferring electrons to the Rieske-type ferredoxin or substituting for the reductase in the alkene monooxygenase complex. Likewise, other bacterial and plant ferredoxins were unable to substitute for the Rieske-type ferredoxin in mediating electron transfer to the oxygenase. The biochemical properties of alkene monooxygenase described in this study suggest that this enzyme combines elements of both the well-characterized aromatic dioxygenase (two-component electron transfer scheme) and methane monooxygenase (small regulatory protein and diiron oxygenase) multicomponent enzyme systems.
来自黄杆菌属菌株Py2的烯烃单加氧酶是一种诱导酶，它催化短链（C2至C6）烯烃在O2和NADH依赖下环氧化为相应的环氧化物，这是利用脂肪族烯烃作为碳源和能源的起始步骤。在本研究中，烯烃单加氧酶已从无细胞提取物的可溶部分分离为四个组分，每个组分均已纯化至同质，它们是烯烃环氧化活性所必需的。这四个必需组分是：1）一种35.5 kDa的单体蛋白，含有1摩尔FAD和一个可能的2Fe-2S中心；2）一种13.3 kDa的铁氧还蛋白，含有一个Rieske型2Fe-2S簇；3）一种11 kDa的单体蛋白，不含可检测到的辅因子；4）一种212 kDa的α2β2γ2多聚体蛋白，含有四个非血红素铁原子。35.5 kDa的蛋白已被鉴定为NADH还原酶。该还原酶的生理电子受体是Rieske型铁氧还蛋白，它被认为是该系统中还原酶和末端催化组分之间的中间电子载体。212 kDa的蛋白在无细胞提取物中被基于机制的失活剂丙炔特异性失活，这表明它是催化组分，并且含有O2活化和烯烃环氧化的活性位点。该组分的亚基结构和金属分析表明它含有两个双铁中心，每个αβγ原聚体单元一个。无法为11 kDa的蛋白赋予特定的酶活性，但该组分是稳态烯烃环氧化所必需的。烯烃单加氧酶组分在黄杆菌Py2在脂肪族烯烃或环氧化物上生长期间表达，而在其他碳源上生长期间受到抑制。烯烃单加氧酶的电子传递组分具有高度特异性：黄杆菌中存在的其他还原酶活性无法将电子转移到Rieske型铁氧还蛋白或替代烯烃单加氧酶复合物中的还原酶。同样，其他细菌和植物铁氧还蛋白也无法替代Rieske型铁氧还蛋白来介导向加氧酶的电子转移。本研究中描述的烯烃单加氧酶的生化特性表明，这种酶结合了特征明确的芳香族双加氧酶（双组分电子传递方案）和甲烷单加氧酶（小调节蛋白和双铁加氧酶）多组分酶系统的元素。
