Cloning, expression, and sequence analysis of the Bacillus methanolicus C1 methanol dehydrogenase gene

Abstract

The gene (mdh) Bacillus methanolicus Cl is a representative of the thermotolerant methanol-utilizing Bacillus spp. which oxidize methanol by use of an NAD-dependent methanol dehydrogenase (MDH) (3,5,9). The previously reported N-terminal amino acid sequence (27) has shown that the enzyme belongs to a novel family of NAD-dependent alcohol dehydrogenases (ADHs) which includes Zymomonas mobilis ADH2 (8), Saccharomyces cerevisiae ADH4 (29), Escherichia coli L-1,2-propanediol oxidoreductase (POR) (7), and the Clostridium acetobutylicum NADPH-dependent butanol dehydrogenase (ADH1) (31). This family of enzymes is different from the horse liver-type ADHs (long chain, type I) or Drosophila-type enzymes (short chain, type II) and will therefore be referred to as type III ADHs.The B. methanolicus MDH is a decameric enzyme with a subunit Mr of 43,000. Each subunit contains a noncovalently but tightly bound NAD(H) cofactor molecule, one zinc atom, and one or two magnesium atoms (27). The S. cerevisiae ADH4 enzyme also has been reported to contain zinc (29), while iron has been detected in the Z. mobilis ADH2 protein (8). The E. coli POR enzyme is stimulated by ferrous ions (25). Further data about the presence of metals in type III ADHs is lacking, and it remains to be decided whether the presence of magnesium in the Bacillus MDH is unique.The recently reported involvement of an activator protein, which stimulates NAD-dependent ADH activity of the purified MDH enzyme, certainly seems unique (4). In this respect, it is significant that in the other members of the type III ADH enzymes, the oxidative reaction does not play a metabolic role: all support a fermentative metabolic function in the respective wild-type organisms. Metabolic dependence on methanol requires a high flux of methanol oxidation: approximately twice as much carbon must be processed by this reaction compared with ethanol utilization. The activator protein has been suggested to play a dedicated role in the release of reducing equivalents from the bound NAD(H) cofactor (1). The occurrence of the activator protein in all methylotrophic Bacillus species investigated (4, 5) may thus form an interesting adaptation to increase the * Corresponding author. reaction rate of NAD-dependent ADHs in the oxidative direction.In this report, the cloning and nucleotide sequence analysis of the B. methanolicus Cl mdh gene are presented. The deduced amino acid sequence was found to share sipificant identity with the other known members of type III ADHs. The C-terminal part of the bifunctional E. coli fermentative ADH (10) could also be incorporated in this cluster. Sequence patterns, which may be related to the distinctive properties of these enzymes, such as NAD(P) and metal binding, have been analyzed and are discussed. Expression and properties of the B. methanolicus MDH in the E. coli host were investigated. MATERIALS AND METHODS

Molecular characterization of two Clostridium acetobutylicum ATCC 824 butanol dehydrogenase isozyme genes

“…No such regions were found in either of the BDHs, and only one such region (6) has yet been identified in any of the enzymes in this new ADH class, despite the fact that all of the proteins with the exception of the C. acetobutylicum ADH have been shown to be able to utilize NADH as a cofactor (6,7,10,33,35,43).…”

Evolution of an Escherichia coli Protein with Increased Resistance to Oxidative Stress

“…It is remarkable that the relatively conservative hydrophobic amino acid substitutions, Ile 7 3 Leu and/or Leu 8 3 Val, near the NAD-binding sequence Gly 15 -Arg-Gly-Ala-Val-Gly 20 of FucO (15,16,47) could confer significant protective effects against MCO damage. This resistance, however, is achieved at a price of decrease in protein stability.…”

Identification and functional characterization of a gene for the methanol : N,N'-dimethyl-4-nitrosoaniline oxidoreductase from Mycobacterium sp. strain JC1 (DSM 3803)

“…On the other hand, very little is known about the physiological role and regulation of the genes for the key enzymes responsible for the oxidation of methanol in Gram-positive methylotrophic bacteria. Only the genes for NAD-dependent MDHs of B. methanolicus strains C1 (de Vries et al, 1992) and MGA3 (Brautaset et al, 2004) have been cloned and sequenced, and several genes, including the gene for MDH, involved in the methylotrophy of B. methanolicus MGA3 were identified to be transcriptionally upregulated in cells growing with methanol (Jakobsen et al, 2006).…”