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. Jörnvall, 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.