The L-lactate dehydrogenase gene of the hyperthermophilic bacterium Thermotoga maritima cloned by complementation in Escherichia coli.
The gene for a L(+)-lactate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was cloned by complementation of an Escherichia coli pfl. Idh mutant. The gene is part of a 4.5 kb SauIIIA fragment obtained by partial digestion of the Thermotoga genome. The DNA fragment was physically mapped and the putative Shine-Dalgarno sequence within the non-coding region determined. The gene contains 960 bp, including the stop codon, corresponding to 319 amino acids/subunit of the homotetrameric enzyme. Part of the amino acid sequence was confirmed by Edman degradation of peptides obtained from nanomolar quantities of the purified enzyme by tryptic digestion. A comparison of the amino acid sequence with those of known prokaryotic L-lactate dehydrogenases reveals a high similarity, especially with the enzyme from thermophilic sources, where up to 48% identity is found. The gene was expressed as an active enzyme in a heterologous host.
Ostendorp
R
R
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Germany.
Liebl
W
W
Schurig
H
H
Jaenicke
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D005816:Genetic Complementation Test; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology* / Q000235:genetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
D004269:DNA, Bacterial; D007770:L-Lactate Dehydrogenase