Clonorchis sinensis: molecular cloning and functional expression of novel cytosolic malate dehydrogenase.
The NAD-dependent cytosolic malate dehydrogenase (cMDH, EC 1.1.1.37) plays a pivotal role in the malate-aspartate shuttle pathway that operates in a metabolic coordination between cytosol and mitochondria, and thus is crucial for the survival and pathogenicity of the parasite. In the high throughput sequencing of the cDNA library constructed from the adult stage of Clonorchis sinensis, a cDNA clone containing 1152bp insert was identified to encode a putative peptide of 329 amino acids possessing more than 50% amino acid sequence identities with the cMDHs from other organisms such as fish, plant, and mammal. But low sequence similarities have been found between this cMDH and mitochondrial malate dehydrogenase as well as glyoxysomal malate dehydrogenase from other organisms. Northern blot analysis showed the size of the C. sinensis cMDH mRNA was 1.2 kb. The cMDH was expressed in Escherichia coli M15 as a His-tag fusion protein and purified by BD TALON metal affinity column. The recombinant cMDH showed high MDH activity of 241 U mg(-1), without lactate dehydrogenase and NADP(H) selectivity. It provides a model for the structure, function analysis, and drug screening on cMDH.
Zheng
Nancai
N
Department of Parasitology, Preclinical School of Sun Yat-sen University, Guangzhou 510089, PR China.
Xu
Jin
J
Wu
Zhongdao
Z
Chen
Jinzhong
J
Hu
Xuchu
X
Song
Linxia
L
Yang
Guang
G
Ji
Chaoneng
C
Chen
Shouyi
S
Gu
Shaohua
S
Ying
Kang
K
Yu
Xinbing
X
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D003003:Clonorchiasis / Q000469:parasitology; D003004:Clonorchis sinensis / Q000201:enzymology / Q000235:genetics*; D016384:Consensus Sequence; D003600:Cytosol / Q000201:enzymology; D018509:DNA, Helminth / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005399:Fishes; D015971:Gene Expression Regulation, Enzymologic; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D018510:RNA, Helminth / Q000737:chemistry; D011817:Rabbits
D018509:DNA, Helminth; D018510:RNA, Helminth; D008291:Malate Dehydrogenase