Ma K, Adams M W
Department of Biochemistry and Molecular Biology and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602, USA.
J Bacteriol. 1999 Feb;181(4):1163-70. doi: 10.1128/JB.181.4.1163-1170.1999.
Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at 100 degreesC by the fermentation of peptides and carbohydrates to produce acetate, CO2, and H2, together with minor amounts of ethanol. The organism also generates H2S in the presence of elemental sulfur (S0). Cell extracts contained NADP-dependent alcohol dehydrogenase activity (0.2 to 0.5 U/mg) with ethanol as the substrate, the specific activity of which was comparable in cells grown with and without S0. The enzyme was purified by multistep column chromatography. It has a subunit molecular weight of 48,000 +/- 1,000, appears to be a homohexamer, and contains iron ( approximately 1.0 g-atom/subunit) and zinc ( approximately 1.0 g-atom/subunit) as determined by chemical analysis and plasma emission spectroscopy. Neither other metals nor acid-labile sulfur was detected. Analysis using electron paramagnetic resonance spectroscopy indicated that the iron was present as low-spin Fe(II). The enzyme is oxygen sensitive and has a half-life in air of about 1 h at 23 degreesC. It is stable under anaerobic conditions even at high temperature, with half-lives at 85 and 95 degreesC of 160 and 7 h, respectively. The optimum pH for ethanol oxidation was between 9. 4 and 10.2 (at 80 degreesC), and the apparent Kms (at 80 degreesC) for ethanol, acetaldehyde, NADP, and NAD were 29.4, 0.17, 0.071, and 20 mM, respectively. P. furiosus alcohol dehydrogenase utilizes a range of alcohols and aldehydes, including ethanol, 2-phenylethanol, tryptophol, 1,3-propanediol, acetaldehyde, phenylacetaldehyde, and methyl glyoxal. Kinetic analyses indicated a marked preference for catalyzing aldehyde reduction with NADPH as the electron donor. Accordingly, the proposed physiological role of this unusual alcohol dehydrogenase is in the production of alcohols. This reaction simultaneously disposes of excess reducing equivalents and removes toxic aldehydes, both of which are products of fermentation.
激烈火球菌是一种嗜热古菌，在100摄氏度时生长最佳，通过肽和碳水化合物发酵产生乙酸盐、二氧化碳和氢气，同时产生少量乙醇。该生物体在元素硫（S0）存在的情况下还会产生硫化氢。细胞提取物含有以乙醇为底物的NADP依赖性乙醇脱氢酶活性（0.2至0.5 U/mg），其比活性在有或没有S0的情况下生长的细胞中相当。该酶通过多步柱色谱法纯化。它的亚基分子量为48,000±1,000，似乎是同六聚体，通过化学分析和等离子体发射光谱法测定含有铁（约1.0克原子/亚基）和锌（约1.0克原子/亚基）。未检测到其他金属和酸不稳定硫。使用电子顺磁共振光谱分析表明铁以低自旋Fe(II)形式存在。该酶对氧气敏感，在23摄氏度空气中的半衰期约为1小时。即使在高温下，它在厌氧条件下也很稳定，在85和95摄氏度下的半衰期分别为160和7小时。乙醇氧化的最佳pH值在9.4至10.2之间（80摄氏度），乙醇、乙醛、NADP和NAD的表观Km值（80摄氏度）分别为29.4、0.17、0.071和20 mM。激烈火球菌乙醇脱氢酶利用一系列醇类和醛类，包括乙醇、2-苯乙醇、色醇、1,3-丙二醇、乙醛、苯乙醛和甲基乙二醛。动力学分析表明，以NADPH作为电子供体时，该酶明显更倾向于催化醛的还原。因此，这种不寻常的乙醇脱氢酶的生理作用被认为是参与醇类的产生。该反应同时处理了过量的还原当量并去除了有毒醛类，这两者都是发酵的产物。
