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.
Yang
Guang
G
Department of Parasitology, Medical School, Jinan University, Guangzhou 510632, People's Republic of China.
Jing
Chunxia
C
Zhu
Peixian
P
Hu
Xuchu
X
Xu
Jin
J
Wu
Zhongdao
Z
Yu
Xinbing
X
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002264:Carboxylic Acids / Q000378:metabolism; D003004:Clonorchis sinensis / Q000201:enzymology / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006072:Gossypol / Q000494:pharmacology; D015801:Helminth Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
D002264:Carboxylic Acids; D004791:Enzyme Inhibitors; D015801:Helminth Proteins; D007770:L-Lactate Dehydrogenase; D006072:Gossypol