Cloning and overexpression of Lactobacillus helveticus D-lactate dehydrogenase gene in Escherichia coli.
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.
Kochhar
S
S
Nestlé Research Centre, Vers-chez-les-Blanc, Lausanne, Switzerland.
Hottinger
H
H
Chuard
N
N
Taylor
P G
PG
Atkinson
T
T
Scawen
M D
MD
Nicholls
D J
DJ
D000429:Alcohol Oxidoreductases / Q000737:chemistry; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007770:L-Lactate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D045022:Lactate Dehydrogenases; D007778:Lactobacillus / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D015183:Restriction Mapping; D016415:Sequence Alignment
D004269:DNA, Bacterial; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; D045022:Lactate Dehydrogenases; C047961:2-hydroxyisocaproate dehydrogenase; D007770:L-Lactate Dehydrogenase; C071195:D-lactate dehydrogenase