Ismaiel A A, Zhu C X, Colby G D, Chen J S
Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg 24061.
J Bacteriol. 1993 Aug;175(16):5097-105. doi: 10.1128/jb.175.16.5097-5105.1993.
Two primary alcohols (1-butanol and ethanol) are major fermentation products of several clostridial species. In addition to these two alcohols, the secondary alcohol 2-propanol is produced to a concentration of about 100 mM by some strains of Clostridium beijerinckii. An alcohol dehydrogenase (ADH) has been purified to homogeneity from two strains (NRRL B593 and NESTE 255) of 2-propanol-producing C. beijerinckii. When exposed to air, the purified ADH was stable, whereas the partially purified ADH was inactivated. The ADHs from the two strains had similar structural and kinetic properties. Each had a native M(r) of between 90,000 and 100,000 and a subunit M(r) of between 38,000 and 40,000. The ADHs were NADP(H) dependent, but a low level of NAD(+)-linked activity was detected. They were equally active in reducing aldehydes and 2-ketones, but a much lower oxidizing activity was obtained with primary alcohols than with secondary alcohols. The kcat/Km value for the alcohol-forming reaction appears to be a function of the size of the larger alkyl substituent on the carbonyl group. ADH activities measured in the presence of both acetone and butyraldehyde did not exceed activities measured with either substrate present alone, indicating a common active site for both substrates. There was no similarity in the N-terminal amino acid sequence between that of the ADH and those of fungi and several other bacteria. However, the N-terminal sequence had 67% identity with those of two other anaerobes, Thermoanaerobium brockii and Methanobacterium palustre. Furthermore, conserved glycine and tryptophan residues are present in ADHs of these three anaerobic bacteria and ADHs of mammals and green plants.
两种伯醇（1-丁醇和乙醇）是几种梭菌属物种的主要发酵产物。除了这两种醇类，仲醇2-丙醇由拜氏梭菌的一些菌株产生，浓度约为100 mM。已从产2-丙醇的拜氏梭菌的两个菌株（NRRL B593和NESTE 255）中纯化得到一种醇脱氢酶（ADH）并使其达到同质。暴露于空气中时，纯化后的ADH是稳定的，而部分纯化的ADH则会失活。这两个菌株的ADH具有相似的结构和动力学特性。每个ADH的天然分子量在90,000至100,000之间，亚基分子量在38,000至40,000之间。这些ADH依赖于NADP(H)，但检测到低水平的NAD(+)连接活性。它们在还原醛类和2-酮类方面具有同等活性，但伯醇的氧化活性远低于仲醇。醇形成反应的kcat/Km值似乎是羰基上较大烷基取代基大小的函数。在丙酮和丁醛同时存在的情况下测得的ADH活性不超过单独存在任何一种底物时测得的活性，这表明两种底物具有共同的活性位点。ADH的N端氨基酸序列与真菌和其他几种细菌的N端氨基酸序列没有相似性。然而，其N端序列与另外两种厌氧菌——布氏嗜热厌氧菌和沼泽甲烷杆菌的N端序列有67%的同一性。此外，这三种厌氧菌的ADH以及哺乳动物和绿色植物的ADH中都存在保守的甘氨酸和色氨酸残基。
