Shimizu Yasuhiro, Sakuraba Haruhiko, Kawakami Ryushi, Goda Shuichiro, Kawarabayasi Yutaka, Ohshima Toshihisa
Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, 2-1 Minamijosanjimacho, Tokushima, Japan.
Extremophiles. 2005 Aug;9(4):317-24. doi: 10.1007/s00792-005-0447-2. Epub 2005 May 18.
A gene encoding the L-threonine dehydrogenase homologue has been identified in a hyperthermophlic archaeon Pyrococcus horikoshii OT3 via genome sequencing. The gene was cloned and expressed in Escherichia coli. The purified enzyme from the recombinant E. coli was extremely thermostable; the activity was not lost after incubation at 100 degrees C for 20 min. The enzyme (molecular mass: 192 kDa) is composed of a tetrameric structure with a type of subunit (41 kDa). The enzyme is specific for NAD and utilizes L-threonine, L-serine and DL-threo-3-phenylserine as the substrate. The enzyme required divalent cations such as Zn(2+), Mn(2+) and Co(2+) for the activity, and contained one zinc ion/subunit. The K(m) values for L-threonine and NAD at 50 degrees C were 0.20 mM and 0.024 mM, respectively. Kinetic analyses indicated that the L-threonine oxidation reaction proceeds via a random mechanism with regard to the binding of L-threonine and NAD. The enzyme showed pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of NADH. This is the first description of the characteristics of an L-threonine dehydrogenase from the archaea domain.
通过基因组测序，在嗜热古菌火之神栖热袍菌OT3中鉴定出一个编码L-苏氨酸脱氢酶同源物的基因。该基因被克隆并在大肠杆菌中表达。从重组大肠杆菌中纯化得到的酶具有极高的热稳定性；在100℃孵育20分钟后活性并未丧失。该酶（分子量：192 kDa）由一种亚基（41 kDa）组成的四聚体结构。该酶对NAD具有特异性，并利用L-苏氨酸、L-丝氨酸和DL-苏式-3-苯基丝氨酸作为底物。该酶的活性需要二价阳离子如Zn(2+)、Mn(2+)和Co(2+)，且每个亚基含有一个锌离子。在50℃时，L-苏氨酸和NAD的K(m)值分别为0.20 mM和0.024 mM。动力学分析表明，L-苏氨酸氧化反应在L-苏氨酸和NAD的结合方面遵循随机机制。该酶对NADH烟酰胺部分C4位的氢转移表现出前-R立体特异性。这是首次对来自古菌域的L-苏氨酸脱氢酶的特性进行描述。
