Molecular genetic characterization of the thermostable L-lactate dehydrogenase gene (ldhL) of Thermoanaerobacter ethanolicus JW200 and biochemical characterization of the enzyme.
The structural gene ldhL for a thermostable L-(+)-lactate dehydrogenase was cloned from Thermoanaerobacter ethanolicus JW200. The nucleotide sequence of the ldhL gene was determined and shown to have the capacity to encode a protein of 311 amino acids (33.5 kDa). By 5'-RACE analysis, the ldhL transcription start point was confirmed to be derived from the -10 region closest to the initiation codon. The enzyme was overexpressed in Escherichia coli and purified to homogeneity by nickel-affinity chromatography. It was shown to be allosteric in the presence of fructose-1,6-bisphosphate. The optimum pH and temperature for the enzyme were 5.8 and 60 degrees capital ES, Cyrillic in the pyruvate reduction and 7.0 and 70 degrees capital ES, Cyrillic in the lactate oxidation reaction, respectively. The kinetic parameters K(m,app) and k(cat,app) for pyruvate were 0.18 mM and 520 U/mg, respectively, and in the absence of fructose-1,6-bisphosphate, a 1.2-fold increase in K(m,app) and a 16-fold decrease in k(cat,app) were determined. The K(m,app) and k(cat,app) values for lactate were 60 mM and 0.58 U/mg, respectively, and they were not affected by fructose-1,6-bisphosphate. The enzyme was greatly inhibited by Zn2+, Ag(+), Cu2+, Fe3+, and Pb(3+). The extreme thermostability of the enzyme was reflected in its unaltered activity over 5 h at 70 degrees C.
Zhou
Q
Q
Jiangsu Key Laboratory for Biodiversity and Bio-resources, College of Life Sciences, Nanjing Normal University, Nanjing, 210046, China. qqzhouqing@hotmail.com
Shao
W-L
WL
D000494:Allosteric Regulation; D001483:Base Sequence; D006597:Fructose-Bisphosphatase / Q000378:metabolism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D046968:Thermoanaerobacter / Q000201:enzymology*
D011994:Recombinant Proteins; D007770:L-Lactate Dehydrogenase; D006597:Fructose-Bisphosphatase