The primary structure of Hyphomicrobium X dimethylamine dehydrogenase. Relationship to trimethylamine dehydrogenase and implications for substrate recognition.
The gene encoding dimethylamine dehydrogenase from Hyphomicrobium X has been cloned and over-expressed in Escherichia coli. Using the chemically determined protein sequence, primers were designed to amplify DNA fragments encoding the proximal and distal parts of the gene. These fragments were used to synthesise two probes and the dmd gene was cloned as part of two BamHI fragments isolated from digested genomic DNA. The sequence of the complete open reading frame was determined on both strands and contained 2211 bp coding for a protein of 736 amino acids, including the N-terminal methionine residue that is removed when expressed in the native host. The molecular mass of the processed apoprotein predicted from the DNA sequence is 82,523 Da. Dimethylamine dehydrogenase is closely related to the trimethylamine dehydrogenase of Methylophilus methylotrophus W3A1 (63.5% identical) and other class I FMN-binding beta 8 alpha 8 barrel flavoproteins. Residues in the active site of trimethylamine dehydrogenase that are known, or implicated, to be important in catalysis are conserved in dimethylamine dehydrogenase. Sequence alignment of dimethylamine and trimethylamine dehydrogenases suggests that the specificity for secondary and tertiary amines resides in a single amino acid substitution in a substrate-binding aromatic bowl located in the active site of the enzymes.
Yang
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CC
Department of Biochemistry, University of Cambridge, England.
Packman
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LC
Scrutton
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NS
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase; C020064:dimethylamine dehydrogenase