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.
The ubiquitin-proteasome system is an essential mechanism for protein degradation in eukaryotes. Protein ubiquitination is composed of a series of enzymatic reactions. The ubiquitin-conjugating enzyme (E2) is one of the important enzymes involved in the process. A cDNA encoding an E2 enzyme was cloned from a Clonorchis sinensis cDNA library by large-scale sequencing. This new cDNA contains 862 bp with a putative open reading frame of 156 amino acids. The deduced amino acid sequence is 77% identical to the human E2, HHR6A and HHR6B. The coding region of this cDNA was expressed in E. coli as a GST-tagged protein, and was purified to electrophoretic homogeneity. Enzymatic assays showed that this E2 had the capacity to form a thiolester linkage, and could conjugate ubiquitin to histone H2A in an E3-independent manner in vitro, which indicated that the expressed protein was functionally active. The nucleotide sequence reported in this paper has been submitted to the Genbank Database with accession number AY632078.
The NAD-dependent mitochondrial malate dehydrogenase (mMDH, EC1.1.1.37) plays pivotal roles in tricarboxylic acid and is crucial for the survival and pathogenecity of parasites. A cDNA, which was identified by high throughput sequencing from the cDNA library constructed from adult Clonorchis sinensis, encoded a putative peptide of 341 amino acids with more than 50% identity with mMDHs from other organisms. The mMDH was expressed in Escherichia coli as the recombinant protein with a GST tag and purified by glutathione-Sepharose 4B column. The recombinant mMDH showed MDH activity of 63.6 U/mg, without lactate dehydrogenase activity and NADPH selectivity. The kinetic constants of recombinant mMDH were determined.
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