来自嗜热细菌海栖热袍菌的葡萄糖-6-磷酸脱氢酶：g6pd基因的表达及一种极端嗜热酶的特性分析

Glucose-6-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima: expression of the g6pd gene and characterization of an extremely thermophilic enzyme.

作者信息

Hansen Thomas, Schlichting Bettina, Schönheit Peter

机构信息

Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, Am Botanischen Garten 1-9, D-24118, Kiel, Germany.

出版信息

FEMS Microbiol Lett. 2002 Nov 5;216(2):249-53. doi: 10.1111/j.1574-6968.2002.tb11443.x.

DOI:10.1111/j.1574-6968.2002.tb11443.x

PMID:12435510

Abstract

The gene (open reading frame Tm1155, g6pd) encoding glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) of the hyperthermophilic bacterium Thermotoga maritima was cloned and functionally expressed in Escherichia coli. The purified recombinant enzyme is a homodimer with an apparent molecular mass of 95 kDa composed of 60-kDa subunits. Rate dependence (at 80 degrees C) on glucose-6-phosphate and NADP(+) followed Michaelis-Menten kinetics with apparent K(m) values of 0.15 mM and 0.03 mM, respectively; apparent V(max) values were about 20 U mg(-1). The enzyme also reduced NAD(+) (apparent K(m) 12 mM, V(max) 12 U mg(-1)). The 1000-fold higher catalytic activity (k(cat)/K(m)) with NADP(+) over NAD(+) defines the G6PD as NADP(+) specific in vivo. G6PD activity was competitively inhibited by NADPH with a K(i) value of 0.11 mM. With a temperature optimum of 92 degrees C the enzyme is the most thermoactive G6PD described.

摘要

嗜热栖热菌葡萄糖-6-磷酸脱氢酶（G6PD，EC 1.1.1.49）的编码基因（开放阅读框Tm1155，g6pd）被克隆，并在大肠杆菌中实现了功能表达。纯化后的重组酶是一种同型二聚体，表观分子量为95 kDa，由60 kDa的亚基组成。在80℃下，酶对葡萄糖-6-磷酸和NADP(+)的速率依赖性符合米氏动力学，表观K(m)值分别为0.15 mM和0.03 mM；表观V(max)值约为20 U mg(-1)。该酶还能还原NAD(+)（表观K(m) 12 mM，V(max) 12 U mg(-1)）。与NAD(+)相比，该酶对NADP(+)的催化活性（k(cat)/K(m)）高1000倍，这表明该G6PD在体内对NADP(+)具有特异性。G6PD活性受到NADPH的竞争性抑制，K(i)值为0.11 mM。该酶的最适温度为92℃，是已报道中热活性最高的G6PD。