Cloning and overexpression of Lactobacillus helveticus D-lactate dehydrogenase gene in Escherichia coli

Eur J Biochem. 1992 Sep 15;208(3):799-805. doi: 10.1111/j.1432-1033.1992.tb17250.x.


NAD(+)-dependent D-lactate dehydrogenase from Lactobacillus helveticus was purified to apparent homogeneity, and the sequence of the first 36 amino acid residues determined. Using forward and reverse oligonucleotide primers, based on the N-terminal sequence and amino acid residues 220-215 of the Lactobacillus bulgaricus enzyme [Kochhar, S., Hunziker, P. E., Leong-Morgenthaler, P. & Hottinger, H. (1992) J. Biol. Chem. 267, 8499-8513], a 0.6-kbp DNA fragment was amplified from L. helveticus genomic DNA by the polymerase chain reaction. This amplified DNA fragment was used as a probe to identify two recombinant clones containing the D-lactate dehydrogenase gene. Both plasmids overexpressed D-lactate dehydrogenase (greater than 60% total soluble cell protein) and were stable in Escherichia coli, compared to plasmids carrying the L. bulgaricus and Lactobacillus plantarum genes. The entire nucleotide sequence of the L. helveticus D-lactate dehydrogenase gene was determined. The deduced amino acid sequence indicated a polypeptide consisting of 336 amino acid residues, which showed significant amino acid sequence similarity to the recently identified family of D-2-hydroxy-acid dehydrogenases [Kochhar, S., Hunziker, P. E., Leong-Morgenthaler, P. & Hottinger, H. (1992) Biochem. Biophys. Res. Commun. 184, 60-66]. The physicochemical and catalytic properties of recombinant D-lactate dehydrogenase were identical to those of the wild-type enzyme, e.g. alpha 2 dimeric subunit structure, isoelectric pH, Km and Kcat for pyruvate and other 2-oxo-acid substrates. The kinetic profiles of 2-oxo-acid substrates showed some marked differences from that of L-lactate dehydrogenase, suggesting different mechanisms for substrate binding and specificity.

Publication types

  • Comparative Study

MeSH terms

  • Alcohol Oxidoreductases / chemistry
  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular
  • DNA, Bacterial / genetics
  • Escherichia coli / genetics
  • Gene Expression
  • Hydrogen-Ion Concentration
  • Isoelectric Point
  • L-Lactate Dehydrogenase / genetics*
  • L-Lactate Dehydrogenase / isolation & purification
  • L-Lactate Dehydrogenase / metabolism
  • Lactate Dehydrogenases*
  • Lactobacillus / enzymology
  • Lactobacillus / genetics*
  • Molecular Sequence Data
  • Molecular Weight
  • RNA, Messenger / genetics
  • Recombinant Proteins / chemistry
  • Restriction Mapping
  • Sequence Alignment


  • DNA, Bacterial
  • RNA, Messenger
  • Recombinant Proteins
  • Alcohol Oxidoreductases
  • Lactate Dehydrogenases
  • 2-hydroxyisocaproate dehydrogenase
  • L-Lactate Dehydrogenase
  • D-lactate dehydrogenase

Associated data

  • GENBANK/M85175
  • GENBANK/M85176
  • GENBANK/M85177
  • GENBANK/S42403
  • GENBANK/S42404
  • GENBANK/X65179
  • GENBANK/X65180
  • GENBANK/X65181
  • GENBANK/X66723
  • GENBANK/Z11975