Cloning and expression of the gene for hydroxypyruvate reductase (D-glycerate dehydrogenase from an obligate methylotroph Hyphomicrobium methylovorum GM2.

Abstract

The gene encoding hydroxypyruvate reductase, catalyzing the asymmetric reduction of hydroxypyruvate to D-glycerate, and its flanking regions were isolated from a methylotrophic bacterium, Hyphomicrobium methylovorum GM2. Nucleotide sequencing of the recombinant plasmids revealed that the hydroxypyruvate-reductase gene codes for the 322-amino-acid protein with calculated molecular mass 35,726 Da. The sequence was confirmed by sequencing the intact enzyme and peptides obtained by digestion of the enzyme with Achromobacter proteinase I. The amino acid sequence of the enzyme showed similarity to members of the D-isomer-specific 2-hydroxyacid dehydrogenase family. The recombinant plasmid, which was constructed by ligation of the cloned gene and an expression vector pKK223-3, was introduced into Escherichia coli HB101. The recombinant enzyme purified from the transformed E. coli cells was indistinguishable from the enzyme isolated from H. methylovorum GM2 by immunological and enzymological analyses.