Trimethylamine dehydrogenase of bacterium W3A1. Molecular cloning, sequence determination and over-expression of the gene.
The gene encoding trimethylamine dehydrogenase (EC 1.5.99.7) from bacterium W3A1 has been cloned. Using the polymerase chain reaction a 530 bp DNA fragment encoding a distal part of the gene was amplified. Using this fragment of DNA as a probe, a clone was then isolated as a 4.5 kb BamHI fragment and shown to encode residues 34 to 729 of trimethylamine dehydrogenase. The polymerase chain reaction was used also to isolate the DNA encoding the missing N-terminal part of the gene. The complete open reading frame contained 2,190 base pairs coding for the processed protein of 729 amino acids which lacks the N-terminal methionine residue. The high-level expression of the gene in Escherichia coli was achieved by the construction of an expression vector derived from the plasmid pKK223-3. The cloning and sequence analysis described here complete the partial assignment of the amino acid sequence derived from chemical sequence [1] and will now permit the refinement of the crystallographic structure of trimethylamine dehydrogenase and also a detailed investigation of the mechanism and properties of the enzyme by protein engineering.
Boyd
G
G
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110.
Mathews
F S
FS
Packman
L C
LC
Scrutton
N S
NS
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli; D008969:Molecular Sequence Data; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis
D000596:Amino Acids; D003062:Codon; D011994:Recombinant Proteins; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase