Cloning and over‐expression in Escherichia coli of the gene encoding NADPH group III alcohol dehydrogenase from Thermococcus hydrothermalis

Abstract

A NADP-dependent group III alcohol dehydrogenase (ADH) was purified from the hyperthermophilic strictly anaerobic archaeon Thermococcus hydrothermalis, which grows at an optimum temperature of 85 8C and an optimum pH of 6. The gene encoding this enzyme was cloned, sequenced, and over-expressed in Escherichia coli. The recombinant enzyme was purified, characterized and compared with the native form of the enzyme. The enzyme structure is pH-dependent, being a 197-kDa tetramer (subunit of 45 kDa) at pH 10.5, the pH optimum for alcohol oxidation, and a 80.5-kDa dimer at pH 7.5, the pH optimum for aldehyde reduction. The kinetic parameters of the enzyme show that the affinity of the enzyme is greater for the aldehyde substrate and NADPH cofactor, suggesting that the dimeric form of the enzyme is probably the active form in vivo. The ADH of T. hydrothermalis oxidizes a series of primary aliphatic and aromatic alcohols preferentially from C 2 to C 8 but is also active towards methanol and glycerol and stereospecific for monoterpenes. T. hydrothermalis ADH is the first Thermococcale ADH to be cloned and overproduced in a mesophilic heterologous expression system, and the recombinant and the native forms have identical main characteristics.Keywords: alcohol dehydrogenase; NADP-dependent; over-expression; pH-dependent structure; thermophilic.Alcohol dehydrogenases (ADH) are present in all organisms. The NAD-or NADP-dependent dehydrogenases can be divided into three major groups [19]. Only the N-terminal amino acid sequences are available for these latter two ADHs.The genes encoding five archaeal ADHs have been identified: the genes for the two closely related zinc-containing ADHs from Sulfolobus spp. have recently been cloned and over-expressed in E. coli [12], the gene encoding the ironcontaining ADH from T. zilligii [16], and the genes for the two ADHs from P. furiosus [14] have also been cloned and overexpressed in E. coli.An ADH activity was found [20] in crude extracts of the hyperthermophilic archaeon T. hydrothermalis [21] growing at an optimal temperature of 85 8C as part of a search for new ADHs with different catalytic properties involved in bioconversion. This report describes the purification of the native enzyme, the design of an oligonucleotidic probe corresponding to the N-terminal amino acid sequence of the protein, and the cloning of its gene. The recombinant enzyme was overexpressed in E. coli to compare the main characteristics of the native and recombinant enzymes and to define the substrate specificity, regulatory properties and thermostability of the recombinant enzyme. M A T E R I A L S A N D M E T H O D SOrganisms, plasmids and chemicals T. hydrothermalis [21] is a hyperthermophilic archaeon isolated from a chimney wall sample collected on the East Pacific Rise at latitude 218N. This strain is a strictly anaerobic coccus, that grows at an optimum temperature of 85 8C, a pH of 6.0 and a sea salt concentration of 20±40 g´L 21. Elemental sulfur greatly enhances the growth of T. hydrothermalis, but ca...

Thermostable Alcohol Dehydrogenase from Thermococcus kodakarensis KOD1 for Enantioselective Bioconversion of Aromatic Secondary Alcohols

“…These thermostable ADHs include iron-containing ADHs from Thermococcus litoralis (14), Thermococcus sp. strain ES-1 (15,16), Thermococcus zilligii (17), Thermococcus hydrothermalis (18), and Thermotoga hypogea (19); zinc-containing ADHs from Pyrococcus furiosus (20), Sulfolobus solfataricus (21,22), Sulfolobus tokodaii (23), and Thermococcus guaymasensis (24); shortchain ADHs from P. furiosus (25) and Thermococcus sibiricus (26); and an AKR from Thermotoga maritima (27). Despite the importance of aromatic chiral alcohols, such as (R)-and (S)-1-phenylethanols, as useful building blocks in pharmaceutical applications (5,28), only four thermostable ADHs, namely, a zinc-containing ADH from Aeropyrum pernix (29,30), two short-chain ADHs from Sulfolobus acidocaldarius (31,32), and an AKR from P. furiosus (33), have been mentioned to be active toward aromatic ketones.…”

Characterization of a Zinc-Containing Alcohol Dehydrogenase with Stereoselectivity from the Hyperthermophilic Archaeon Thermococcus guaymasensis

“…Among strains of the genus Thermococcus, Thermococcus strain ES1 was first reported to produce ethanol under S 0 -limiting conditions (42), one of the attractive renewable energy resources. Subsequently, native ADHs have been purified and characterized, and all of these ADHs are iron containing and proposed to be responsible for ethanol formation (3,39,42,43).…”

Two novel metal-independent long-chain alkyl alcohol dehydrogenases from Geobacillus thermodenitrificans NG80-2

“…M-1, which exhibited mainly reductive activity on medium-chain alkyl aldehydes (C 2 to C 14 ), with little oxidative activity detected, and was assumed not to participate in the degradation of long-chain alkanes (Tani et al, 2000). On the other hand, both ADH1 and ADH2 are expressed at similar levels (less than 50 % difference) in NG80-2 cells grown with hexadecane and with sucrose, as indicated by 2-DE analysis (Feng et al, 2007); thus they are are expected to be involved in other cellular processes such as aldehyde detoxication and alcohol fermentation, as for other characterized ADHs (Antoine et al, 1999;Daniel et al, 1995;Hirakawa et al, 2004;Hosaka et al, 2001;Larroy et al, 2003;Ma et al, 1994Ma et al, , 1995Ma & Adams, 1999;Scopes, 1983;Tani et al, 2000;Ying et al, 2007). It is likely that the major activity of ADH1 and ADH2 is diverted to oxidation of long-chain alcohols when long-chain alkanes are utilized as carbon and energy sources.…”