THE ALCALIGENES-EUTROPHUS-LDH STRUCTURAL GENE ENCODES A NOVEL TYPE OFLACTATE-DEHYDROGENASE

The lactate dehydrogenase gene, ldh, of Alcaligenes eutrophus H16 was identified on a 14-kbp EcoRI restriction fragment of a genomic library in the cosmid pHC79 by hybridization with a 50-mer synthetic oligonuc leotide which was derived from the N-terminal amino acid sequence of t he purified enzyme. Recombinant strains of Escherichia coli JM83, whic h harboured a 2.0-kbp PstI subfragment in pUC9-1, expressed LDH at a h igh level, if ldh was downstream from and colinear to the E. coli lac promoter. The nucleotide sequence of a region of 4245 bp revealed seve ral open reading frames which might represent coding regions. One repr esented the ldh gene. The amino acid sequence deduced from ldh exhibit ed 29% and 36% identity to the L-malate dehydrogenase of Methanothermu s fervidus and to the putative translation product of an E. coli seque nce of unknown function, respectively. The ldh was separated by short intergenic regions from two other open reading frames: ORF5 was locate d downstream of and colinear to ldh, and its putative translational pr oduct revealed 38 to 56% amino acid identity to penicillin-binding pro teins. ORF3 was located upstream of and colinear to ldh, and its putat ive gene translational product represented a hydrophobic protein. A se quence, which resembled the A. eutrophus alcohol dehydrogenase promote r, was detected upstream of ORF3, which most probably represents the f irst transcribed gene of an operon consisting of ORF3, ldh and ORF5.