Molecular cloning and characterization of a novel lactate dehydrogenase gene from Clonorchis sinensis.

From a Clonorchis sinensis adult worm cDNA library, we isolated a cDNA clone encoding a novel lactate dehydrogenase (LDH) gene which encoded a putative protein with a predicted molecular weight of 35.6 kDa. The optimum pH and temperature for the enzyme were 7.5 and 50 degrees C in the pyruvate reduction while 11 and 80 degrees C in the lactate oxidation reaction, respectively. CsLDH showed no substrate inhibition by high lactate and NAD(+) concentration, and the optimal pyruvate and optimal NADH concentrations were 10 and 0.5 mmol/l, respectively. The relative activities of these 2-oxocarboxylic acids were pyruvic acid>2-ketobutyrate>oxalacetic acid>alpha-ketoglutaric acid>phenylpyruvate. The cofactor 3-acetylpyridine adenine dinucleotide was much more effective than NAD(+). The cofactor analogs in which the nicotinamide ring is replaced by 3-pyridinealdehyde were lower activity cofactors, while the nicotinamide ring is replaced by nicotinic acid or thionicotinamide which is not a cofactor to CsLDH. The succinic acid and malic acid are not substrates of CsLDH. Cu(2+), Fe(2+), and Zn(2+) greatly inhibited the CsLDH activity both in the direction of pyruvate reduction and in the direction of lactate oxidation. The inhibition of CsLDH by gossypol may make gossypol a potential therapy drug or a lead compound for C. sinensis. Accordingly, the CsLDH may be a novel potential drug target.