Cloning, nucleotide sequencing, and expression of an opine dehydrogenase gene from Arthrobacter sp. strain 1C

The gene coding for opine dehydrogenase from Arthrobacter sp. strain 1C was cloned onto plasmid pBlue-script KS(2), and the nucleotide sequence of the 1,077-bp open reading frame consisting of 359 codons was identified as the odh gene. Transformed Escherichia coli cells overproduced NAD1-dependent opine dehydro-genase under control of the promoter of the lac gene on pBluescript KS(2). We purified and characterized a new enzyme, NAD1-de-pendent opine dehydrogenase {ODH; N-[1-D-(carboxyl)ethyl]-L-phenylalanine:NAD1 oxidoreductase (L-phenylalanine form-ing) (EC 1.5.1.—)} from a soil isolate of Arthrobacter sp. strain 1C (3). The enzyme has a molecular weight of about 70,000 and consists of two identical subunits with a molecular weight of about 36,000 (each). The enzyme catalyzes a reversible oxidation-reduction reaction of opine-type secondary amine dicarboxylic acids. In the oxidative deamination reaction, the enzyme is active toward opines, such as N-[1-D-(carboxyl) ethyl]-L-methionine (methiopine) (10) and N-[1-D-(carboxyl) ethyl]-L-phenylalanine. In the reductive secondary amine-forming reactions with NADH as a cofactor, the enzyme uti-lizes hydrophobic L-amino acids, such as L-methionine, L-iso