Identification of a Magnesium-dependent NAD(P)(H)-binding Domain in the Nicotinoprotein Methanol Dehydrogenase from Bacillus methanolicus

Harm J Hektor,Harm Kloosterman,Lubbert Dijkhuizen

doi:10.1074/jbc.m207547200

Abstract

The Bacillus methanolicus methanol dehydrogenase (MDH) is a decameric nicotinoprotein alcohol dehydrogenase (family III) with one Zn(2+) ion, one or two Mg(2+) ions, and a tightly bound cofactor NAD(H) per subunit. The Mg(2+) 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.

Highlights

Mutant G95A displayed a rather low methanol dehydrogenase (MDH) activity, whereas mutant S97G was insensitive to activator protein but displayed “fully activated” MDH reaction rates
Methanol dehydrogenase (MDH)1 of Bacillus methanolicus belongs to family III of NAD(P)-dependent alcohol dehydrogenases (ADHs) [2, 3], distinct from the zinc-containing medium chain dehydrogenases/reductases and the zinc-lacking short chain 32 ADHs [4, 5]
The addition of MgSO4 to the growth medium was previously shown to be essential for Mg2ϩ and cofactor NAD(H) binding in MDH protein expressed in E. coli, determining the sensitivity of MDH to the stimulatory effect of activator protein, resulting in hydrolytic removal of the NMN(H) moiety of cofactor NAD(H) [1]

Summary

Introduction

Methanol dehydrogenase (MDH) of Bacillus methanolicus belongs to family III of NAD(P)-dependent alcohol dehydrogenases (ADHs) [2, 3], distinct from the zinc-containing medium chain dehydrogenases/reductases (family I) and the zinc-lacking short chain 32 ADHs (family II) [4, 5]. B. methanolicus MDH requires a second, exogenous NADϩ for methanol oxidation, serving as a coenzyme and resulting in reoxidation of the NADH cofactor [11] These two NAD(H) molecules are not exchanged during the reaction [11]. Activator protein-mediated activation of MDH is characterized by hydrolytic removal of the NMN(H) moiety of cofactor NAD(H) and converts the Ping-Pong type of reaction mechanism of MDH to a ternary complex mechanism, implying direct transfer of electrons from methanol to coenzyme NADϩ [1]. In MDH of B. methanolicus and in most other family III ADHs, only an imperfect fingerprint (G13XG15) is found in the N-terminal part of the protein (Fig. 1A) These enzymes contain strongly modified or novel NAD(P)(H)-binding domains, allowing binding of NADϩ coenzyme in MDH and tight binding of NAD(P)(H) cofactors in MDH and MNO

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Conclusion