Identification and characterization of epoxide carboxylase activity in cell extracts of Nocardia corallina B276.
Identification and characterization of epoxide carboxylase activity in cell extracts of Nocardia corallina B276.
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珊瑚诺卡氏菌 B276 细胞提取物中环氧化物羧化酶活性的鉴定和表征。
DOI：
10.1128/jb.180.8.2072-2078.1998
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发表时间：
1998
影响因子：
3.2
通讯作者：
Ensign,SA
中科院分区：
文献类型：
--
作者：
Allen,JR;Ensign,SA
研究了利用烯烃的放线菌corallinaB276对脂肪族环氧化合物（环氧烷烃）的代谢。悬浮液的探讨。以丙烯为碳源培养的珊瑚细胞容易降解丙烯和环氧丙烷，而葡萄糖培养细胞的悬浮液则不会。在葡萄糖培养的细胞中添加丙烯和环氧丙烷导致丙烯和环氧丙烷降解活性的时间依赖性增加，这被添加利福平和氯霉素所阻止。烯烃和环氧化物降解活性的表达与葡萄糖培养细胞提取物中不存在的几种多肽的高水平表达相关。丙烯生长n细胞悬浮液降解环氧丙烷和环氧丁烷。珊瑚的生长受到二氧化碳增加的刺激，并受到二氧化碳减少的抑制。细胞提取物催化环氧丙烷羧基化生成乙酰乙酸，该反应依赖于CO2、NAD+和还原剂（NADPH或二硫代苏糖醇）的加入。在没有CO2的情况下，细胞提取物将环氧丙烷异构化成丙酮，其速率比环氧丙烷羧化速率低约10倍。甲基环氧丙烷是一种具有时间依赖性、不可逆的环氧烷烃降解失活剂。这些性质证明了环氧烷烃的代谢。corallina通过羧基化反应生成β-酮酸作为产物，并提供证据表明环氧化羧化酶参与该反应，其性质和辅因子需求与革兰氏阴性细菌黄杆菌Py2的四组分环氧化羧化酶系统相似（J. R. Allen和S. a . Ensign， J. Biol）。化学。272:32121-32128,1997)。从黄杆菌Py2中添加环氧化羧化酶组分I至甲基环氧丙烷失活n。珊瑚提取物恢复了环氧化物羧化酶的活性，并从黄杆菌py2中添加了环氧化物羧化酶组分II。珊瑚提取物刺激环氧化物异构酶率达到与纯化的黄杆菌系统观察到的相同水平。针对黄杆菌Py2环氧化羧化酶成分I的抗体与丙烯生长n中的多肽交叉反应。与组分1具有相同分子量的珊瑚提取物，但不与葡萄糖培养的提取物发生交叉反应。总之，这些结果表明，在系统发育上不同的细菌中，存在一种共同的环氧烷烃代谢途径，该途径涉及二氧化碳固定和多组分环氧化物羧化酶系统的活性。
The metabolism of aliphatic epoxides (epoxyalkanes) by the alkene-utilizing actinomyceteNocardia corallinaB276 was investigated. Suspensions ofN. corallinacells grown with propylene as the carbon source readily degraded propylene and epoxypropane, while suspensions of glucose-grown cells did not. The addition of propylene and epoxypropane to glucose-grown cells resulted in a time-dependent increase in propylene- and epoxypropane-degrading activities that was prevented by the addition of rifampin and chloramphenicol. The expression of alkene- and epoxide-degrading activities was correlated with the high-level expression of several polypeptides not present in extracts of glucose-grown cells. Epoxypropane and epoxybutane degradation by propylene-grown cell suspensions ofN. corallinawas stimulated by the addition of CO2and inhibited by the depletion of CO2. Cell extracts catalyzed the carboxylation of epoxypropane to form acetoacetate in a reaction that was dependent on the addition of CO2, NAD+, and a reductant (NADPH or dithiothreitol). In the absence of CO2, epoxypropane was isomerized by cell extracts to form acetone at a rate approximately 10-fold lower than the rate of epoxypropane carboxylation. Methylepoxypropane was found to be a time-dependent, irreversible inactivator of epoxyalkane-degrading activity. These properties demonstrate that epoxyalkane metabolism inN. corallinaoccurs by a carboxylation reaction forming β-keto acids as products and provide evidence for the involvement in this reaction of an epoxide carboxylase with properties and cofactor requirements similar to those of the four-component epoxide carboxylase enzyme system of the gram-negative bacteriumXanthobacterstrain Py2 (J. R. Allen and S. A. Ensign, J. Biol. Chem. 272:32121–32128, 1997). The addition of epoxide carboxylase component I fromXanthobacterstrain Py2 to methylepoxypropane-inactivatedN. corallinaextracts restored epoxide carboxylase activity, and the addition of epoxide carboxylase component II fromXanthobacterPy2 to activeN. corallinaextracts stimulated epoxide isomerase rates to the same levels observed with the purifiedXanthobactersystem. Antibodies raised againstXanthobacterstrain Py2 epoxide carboxylase component I cross-reacted with a polypeptide in propylene-grownN. corallinaextracts with the same molecular weight as component I but did not cross-react with glucose-grown extracts. Together, these results suggest a common pathway of epoxyalkane metabolism for phylogenetically distinct bacteria that involves CO2fixation and the activity of a multicomponent epoxide carboxylase enzyme system.