Ohshima T, Nunoura-Kominato N, Kudome T, Sakuraba H
Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Japan.
Eur J Biochem. 2001 Sep;268(17):4740-7. doi: 10.1046/j.1432-1327.2001.02394.x.
A novel NADH-dependent glyoxylate reductase has been found in a hyperthermophilic archaeon Thermococcus litoralis DSM 5473. This is the first evidence for glyoxylate metabolism and its corresponding enzyme in hyperthermophilic archaea. NADH-dependent glyoxylate reductase was purified approximately 560-fold from a crude extract of the hyperthermophile by five successive column chromatographies and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 76 kDa, and the enzyme consisted of a homodimer with a subunit molecular mass of approximately 37 kDa. The optimum pH and temperature for enzyme activity were approximately 6.5 and 90 degrees C, respectively. The enzyme was extremely thermostable; the activity was stable up to 90 degrees C. The glyoxylate reductase catalyzed the reduction of glyoxylate and hydroxypyruvate, and the relative activity for hydroxypyruvate was approximately one-quarter that of glyoxylate in the presence of NADH as an electron donor. NADPH exhibited rather low activity as an electron donor compared with NADH. The Km values for glyoxylate, hydroxypyruvate, and NADH were determined to be 0.73, 1.3 and 0.067 mM, respectively. The gene encoding the enzyme was cloned and expressed in Escherichia coli. The nucleotide sequence of the glyoxylate reductase gene was determined and found to encode a peptide of 331 amino acids with a calculated relative molecular mass of 36,807. The amino-acid sequence of the T. litoralis enzyme showed high similarity with those of probable dehydrogenases in Pyrococcus horikoshii and P. abyssi. The purification of the enzyme from recombinant E. coli was much simpler compared with that from T. litoralis; only two steps of heat treatment and dye-affinity chromatography were needed.
在嗜热古菌嗜热栖热球菌DSM 5473中发现了一种新型的依赖NADH的乙醛酸还原酶。这是嗜热古菌中乙醛酸代谢及其相应酶的首个证据。通过五次连续柱色谱和制备型PAGE从嗜热菌的粗提物中纯化出依赖NADH的乙醛酸还原酶，纯化倍数约为560倍。纯化酶的分子量估计为76 kDa，该酶由同源二聚体组成，亚基分子量约为37 kDa。酶活性的最适pH和温度分别约为6.5和90℃。该酶具有极高的热稳定性；其活性在高达90℃时仍保持稳定。乙醛酸还原酶催化乙醛酸和羟基丙酮酸的还原，在以NADH作为电子供体时，羟基丙酮酸的相对活性约为乙醛酸的四分之一。与NADH相比，NADPH作为电子供体时活性相当低。乙醛酸、羟基丙酮酸和NADH的Km值分别测定为0.73、1.3和0.067 mM。编码该酶的基因被克隆并在大肠杆菌中表达。测定了乙醛酸还原酶基因的核苷酸序列，发现其编码一个331个氨基酸的肽段，计算相对分子量为36,807。嗜热栖热球菌酶的氨基酸序列与嗜热栖热球菌和深渊嗜热栖热球菌中可能的脱氢酶的氨基酸序列具有高度相似性。与从嗜热栖热球菌中纯化酶相比，从重组大肠杆菌中纯化酶要简单得多；仅需热处理和染料亲和色谱两步。
