CHARACTERIZATION OF THE GENE ENCODING QUINOHEMOPROTEIN ETHANOL DEHYDROGENASE OF COMAMONAS-TESTOSTERONI

The gene encoding quinohaemoprotein ethanol dehydrogenase type I (QH-E DH(I)) from Comamonas testosteroni has been cloned and sequenced. Comp arison of the amino acid sequence deduced from this with that determin ed for the N-terminal amino acid stretch of purified QH-EDH(I), sugges ts that the gene also contains a leader sequence of 31 residues. Based on this information, the molecular mass of the apoenzyme, i,e. the en zyme without the cofactors pyrroloquinoline quinone (PQQ) and haem c, and without the Ca2+, appears to be 73 200 Da. Alignment of the deduce d amino acid sequence to that of other PQQ-containing dehydrogenases s howed that good similarity (up to 43% identity) exists with most of th em, This also showed that the amino acid residues presumed to be invol ved in PQQ and Ca2+ binding and in the typical features of structure a nd catalysis of methanol dehydrogenase, are conserved at the same posi tions in QH-EDH,. The C-terminal part of the protein, containing the h aem c, exhibited some similarity to cytochromes c(553) from cyanobacte ria and algae. Correct processing of the qhedh gene appeared to occur in Escherichia coli strain JM 109 in which the gene was placed under c ontrol of the lac promoter, as judged from a positive reaction with an tibodies raised against authentic QH-EDH(I), the size of the protein, the presence of haem c in it, and the specific activity value obtained after reconstitution with PQQ The qhedh gene seems to form part of an operon which is organized in a way different from that of the genes r equired for methanol oxidation in methylotrophic bacteria.