Alcohol dehydrogenase gene from Alcaligenes eutrophus: subcloning, heterologous expression in Escherichia coli, sequencing, and location of Tn5 insertions

Abstract

The nucleotide sequence of the gene that encodes the fermentative, multifunctional alcohol dehydrogenase (ADH) in Alcaligenes eutrophus, and of adjacent regions on a 1.8-kilobase-pair PstI fragment was determined. From the deduced amino acid sequence, a molecular weight of 38,549 was calculated for the ADH subunit. The amino acid sequence reveals homologies from 22.3 to 26.3% with zinc-containing alcohol dehydrogenases from eucaryotic organisms (Schizosaccharomyces pombe, Zea mays, mouse, horse liver, and human liver). Most of the 22 amino acid residues, which are strictly conserved in this group of ADHs (H. Jornvall, B. Persson, and J. Jeffery, Eur. J. Biochem. 167:195-201, 1987), either were present in the A. eutrophus enzyme or had been substituted by related amino acids. The A. eutrophus adh gene was transcribed in Escherichia coli only under the control of the lac promoter, but was not expressed by its own promoter. A sequence resembling the E. coli consensus promoter DNA sequence did not contain the invariant T, but a G, in the potential -10 region. In the transposon-induced mutants HC1409 and HC1421, which form ADH constitutively, the insertions of Tn5::mob were localized 56 and 66 base pairs, respectively, upstream of the presumptive translation initiation codon. In contrast to the promoter, the A. eutrophus ribosome-binding site with a GGAG Shine-Dalgarno sequence 6 base pairs upstream of the translation initiation codon was accepted by the E. coli translation apparatus. A stable hairpin structure, which may provide a transcription termination signal, is predicted to occur in the mRNA, with its starting point 21 base pairs downstream from the translation termination codon.Recently, we described the cloning of an 11.5-kilobasepair (kbp) EcoRI fragment which encodes the gene for fermentative alcohol dehydrogenase (ADH) from the strict aerobe Alcaligenes eutrophus (44). This enzyme is a tetramer of relative molecular weight 156,000 and consists of four subunits of equal size. The ADH catalyzes the NAD(P)-dependent oxidation of ethanol, 2,3-butanediol, and acetaldehyde and the reduction of acetaldehyde, acetoin, and diacetyl (66). The wild type synthesizes this multifunctional ADH, together with an NAD-linked lactate dehydrogenase, only when the cells are cultivated under conditions of restricted oxygen supply (58). In addition to the ability to evolve molecular hydrogen (45, 71) and to synthesize poly-,-hydroxybutyric acid (71), both enzymes appear to provide a safety valve for the release of excess reducing power in the absence of exogenous hydrogen acceptors such as oxygen or nitrate.Although many primary structures of eucaryotic ADHs have been elaborated (12,40,54), relatively little information exists on the primary structures of procaryotic ADHs (13, 51) and their genes.

Identification and molecular characterization of the gene coding for acetaldehyde dehydrogenase II (acoD) of Alcaligenes eutrophus

“…The molecular data for acoD fit with the N-terminal amino acid sequence obtained for AcDH-II and also with the position of TnS insertions in two mutants of A. eutrophus impaired in the utilization of acetoin as the sole carbon source for growth. The codon usage and a strong bias for G+C in the third codon position of acoD are in good agreement with corresponding data for other genes of A. eutrophus (22,34,36,49 (36). acoD is preceded by its own promoter sequence, which exhibited high homology with the enterobacterial consensus sequence for o4-dependent promoters, and the transcription start site was identified downstream of this promoter.…”

“…2 and 3). The codon usage in the acoD gene was very similar to those reported for the adh gene (22) or the genes of the aco operon (36). The mentioned genes, the bias toward the use of codons with G or C in position 3 was not as extreme for the glutamate codons; GAA was used 13 times, whereas GAG was used 18 times.…”

Characterization of alcohol dehydrogenase genes of derepressible wild-type Alcaligenes eutrophus H16 and constitutive mutants

“…Since three of the first five codons of the adh gene encode for methionine (13), we were not sure about the correct N terminus of the protein (Met-1-Thr-2-Ala-3-Met-4-Met-5-). A functional Shine-Dalgarno sequence was located six nucleotides upstream of the first methionine codon, which led us to the conclusion that this codon represents the start of the gene (13). To determine the N-terminal amino acid sequence of the ADH, the enzyme was purified to homogeneity as described previously (31).…”

“…Recently, we described the cloning and sequencing of the gene coding for the fermentative ADH of A. eutrophus H16 (13,14). Sequence analysis of the 5' upstream adh region revealed two adjoining potential -10 regions that exhibited homologies to the Escherichia coli cr70 consensus sequence (9,22).…”

“…In addition to the ability to produce molecular hydrogen (15,34) and to synthesize poly(P-hydroxybutyric acid) (34), both enzymes appear to provide a safety valve for the release of excess reducing power in the absence of exogenous hydrogen acceptors such as oxygen or nitrate, as indicated by the excretion of ethanol, 2,3-butanediol, and lactate into the medium (33). The fermentative ADH consists of four identical subunits with a molecular weight of 38,549, as calculated from the nucleotide sequence (13). The ADH catalyzes the NAD(P)-dependent oxidation of ethanol, 2,3-butanediol, and acetaldehyde and the reduction of acetaldehyde, acetoin, and diacetyl (31).…”