Identification of a magnesium-dependent NAD(P)(H)-binding domain in the nicotinoprotein methanol dehydrogenase from Bacillus methanolicus
Identification of a magnesium-dependent NAD(P)(H)-binding domain in the nicotinoprotein methanol dehydrogenase from Bacillus methanolicus
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DOI：
10.1074/jbc.m207547200
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发表时间：
2002-12-06
影响因子：
4.8
通讯作者：
Dijkhuizen, L
中科院分区：
文献类型：
--
作者：
Hektor, HJ;Kloosterman, H;Dijkhuizen, L
甲醇芽孢杆菌甲醇脱氢酶（MDH）是一种十聚体烟蛋白醇脱氢酶（III家族），每个亚基具有一个Zn 2+离子、一个或两个Mg 2+离子和一个紧密结合的辅因子NAD（H）。Mg 2+离子对于MDH中辅因子NAD（H）的结合是必需的。A B。methanolicus激活蛋白强烈地刺激相对低的辅酶NAD（+）依赖性MDH活性，包括辅因子NAD（H）的NMN（H）部分的水解去除（Kloosterman，H.，Vrijbloed，J.W.，和Dijkhuizen，L.（2002）J.Biol.Chem.277，34785-34792）。NAD（P）依赖性醇脱氢酶的III家族成员含有三个独特的保守序列基序（结构域A、B和C）。结构域C被认为参与金属结合，而结构域A和B的功能仍然未知。这篇论文提供了证据，结构域A构成（部分）一个新的镁依赖的NAD（P）（H）-结合域。定点突变体D100 N和K103 R缺乏（大部分）结合的辅因子NAD（H），并失去了所有辅酶NAD（+）依赖的MDH活性。突变体G95 A和S97 G的辅酶NAD（H）结合能力均受到损害，但仍保持辅酶NAD（+）依赖的MDH活性。突变体G95 A表现出相当低的MDH活性，而突变体S97 G对激活蛋白不敏感，但表现出“完全激活”的MDH反应速率。这些氨基酸残基在辅酶和/或辅因子NAD（H）结合MDH中的各种作用进行了讨论。
The Bacillus methanolicus methanol dehydrogenase (MDH) is a decameric nicotinoprotein alcohol dehydrogenase (family III) with one Zn2+ ion, one or two Mg2+ ions, and a tightly bound cofactor NAD(H) per subunit. The Mg2+ ions are essential for binding of cofactor NAD(H) in MDH. A B. methanolicus activator protein strongly stimulates the relatively low coenzyme NAD(+)-dependent MDH activity, involving hydrolytic removal of the NMN(H) moiety of cofactor NAD(H) (Kloosterman, H., Vrijbloed, J. W., and Dijkhuizen, L. (2002) J. Biol. Chem. 277, 34785-34792). Members of family III of NAD(P)-dependent alcohol dehydrogenases contain three unique, conserved sequence motifs (domains A, B, and C). Domain C is thought to be involved in metal binding, whereas the functions of domains A and B are still unknown. This paper provides evidence that domain A constitutes (part of) a new magnesium-dependent NAD(P)(H)-binding domain. Site-directed mutants D100N and K103R lacked (most of the) bound cofactor NAD(H) and had lost all coenzyme NAD(+)-dependent MDH activity. Also mutants G95A and S97G were both impaired in cofactor NAD(H) binding but retained coenzyme NAD(+)-dependent MDH activity. Mutant G95A displayed a rather low MDH activity, whereas mutant S97G was insensitive to activator protein but displayed "fully activated" MDH reaction rates. The various roles of these amino acid residues in coenzyme and/or cofactor NAD(H) binding in MDH are discussed.