Characterization of the gene encoding quinohaemoprotein ethanol dehydrogenase of Comamonas testosteroni.
The gene encoding quinohaemoprotein ethanol dehydrogenase type I (QH-EDHI) from Comamonas testosteroni has been cloned and sequenced. Comparison of the amino acid sequence deduced from this with that determined for the N-terminal amino acid stretch of purified QH-EDHI, suggests that the gene also contains a leader sequence of 31 residues. Based on this information, the molecular mass of the apo-enzyme, i.e. the enzyme without the cofactors pyrroloquinoline quinone (PQQ) and haem c, and without the Ca2+, appears to be 73 200 Da. Alignment of the deduced amino acid sequence to that of other PQQ-containing dehydrogenases showed that good similarity (up to 43% identity) exists with most of them. This also showed that the amino acid residues presumed to be involved in PQQ and Ca2+ binding and in the typical features of structure and catalysis of methanol dehydrogenase, are conserved at the same positions in QH-EDHI. The C-terminal part of the protein, containing the haem c, exhibited some similarity to cytochromes C553 from cyanobacteria and algae. Correct processing of the qhedh gene appeared to occur in Escherichia coli strain JM 109 in which the gene was placed under control of the lac promoter, as judged from a positive reaction with antibodies raised against authentic QH-EDHI, 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 required for methanol oxidation in methylotrophic bacteria.
Stoorvogel
J
J
Department of Microbiology and Enzymology, Delft University of Technology, The Netherlands.
Kraayveld
D E
DE
Van Sluis
C A
CA
Jongejan
J A
JA
De Vries
S
S
Duine
J A
JA
D000429:Alcohol Oxidoreductases / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor)