Trimethylamine dehydrogenase of bacterium W3A1 Molecular cloning, sequence determination and over‐expression of the gene

Abstract

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 sequencing (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.