Molecular cloning and characterization of a novel adenylate kinase 3 gene from Clonorchis sinensis.
Adenylate kinase (AK) is a ubiquitous enzyme that contributes to the homeostasis of adenine nucleotides in living cells. AK catalyzes reversible high energy phosphoryl transfer reactions between ATP (or GTP) and AMP to generate ADP (or GDP). From a Clonorchis sinensis adult worm cDNA library, we isolated a cDNA clone encoding a novel AK3 isozyme. The 956 bp cDNA encodes a putative protein of 228 amino acids with a predicted molecular mass of 26.2 kDa. The recombinant CsAK3 protein produced in Escherichia coli can be refolded into a functional protein with AK3 activity. The optimum pH and temperature for the enzyme are 8.5 and 40 degrees C, respectively. The calculated activation energy is 56.04 kJ mol-1. The Km of the CsAK3 for AMP and GTP are 118 microM and 359 microM, respectively. CsAK3 is inhibited by Ap5A (>70% inhibition by 2.0 mM AP5A). Ap5A may be a potential lead compound acting on C. sinensis in which AK3 as a drug target.
Yang
Guang
G
Department of Parasitology, Medical School, Sun Yat-Sen University, 74 Zhongshan 2 Road, 510089, Guangzhou , P.R. China. guangyangphd@163.com
Yu
Xinbing
X
Wu
Zhongdao
Z
Xu
Jin
J
Song
Linxia
L
Zhang
Hongmei
H
Hu
Xuchu
X
Zheng
Nancai
N
Guo
Lingchen
L
Xu
Jian
J
Dai
Jianfeng
J
Ji
Chaoneng
C
Gu
Shaohua
S
Ying
Kang
K
D000263:Adenylate Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003004:Clonorchis sinensis / Q000201:enzymology* / Q000235:genetics; D018076:DNA, Complementary; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013696:Temperature
D018076:DNA, Complementary; D000263:Adenylate Kinase