Toxocara canis: molecular cloning, characterization, expression and comparison of the kinetics of cDNA-derived arginine kinase.
Arginine kinase (AK) is a member of a highly conserved family of phosphagen kinases. We determined the cDNA sequence of Toxocara canis AK, cloned it in pMAL plasmid and expressed it in Escherichia coli as a fusion protein with maltose-binding protein. The protein has a theoretical molecular mass of 45,376 Da and an estimated isoelectric point (pI) of 8.38. Alignment of the cDNA-derived amino acid sequence of T. canis AK with other phosphagen kinase sequences showed high amino acid identity with other nematode AKs, and phylogenetic analysis placed it as a distinct branch within a nematode AK cluster. Analysis of the N-terminus sequence of T. canis AK revealed the presence of a signal targeting peptide presumably targeting this protein to cytosol or endoplasmic reticulum (ER). T. canis AK showed high activity for l-arginine. The kinetic constants (K(m) = 0.12 mM, K(cat) = 29.18, and K(d) = 0.23 mM) and V(max) (43.76 micromolPi/min/mg protein) of T. canis recombinant-AK were determined for the forward reaction. It also exhibited a synergism for substrate binding (K(d)(Arg)/K(m)(Arg)=1.96). Comparison of K(cat)/K(m)(Arg) values in various arginine kinases indicates that T. canis AK has a high catalytic efficiency (248.19s(-1)mM(-1)). The present study contains the first description of arginine kinase in a zoonotic nematode. The determination of T. canis AK and its phosphagen biosynthetic pathway, which is completely different from those in mammalian host tissues, suggests this enzyme as a possible novel chemotherapy target for VLM syndrome in humans.
Wickramasinghe
Susiji
S
Department of Environmental Health Sciences, Kochi Medical School, Oko, Nankoku City, Kochi 783-8505, Japan.
Uda
Kouji
K
Nagataki
Mitsuru
M
Yatawara
Lalani
L
Rajapakse
R P V J
RP
Watanabe
Yoshiya
Y
Suzuki
Tomohiko
T
Agatsuma
Takeshi
T
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001122:Arginine Kinase / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D021621:Imaging, Three-Dimensional; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D021382:Protein Sorting Signals / Q000235:genetics; D018510:RNA, Helminth / Q000235:genetics; D011994:Recombinant Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017166:Toxocara canis / Q000201:enzymology* / Q000235:genetics*
D018076:DNA, Complementary; D021382:Protein Sorting Signals; D018510:RNA, Helminth; D011994:Recombinant Proteins; D001120:Arginine; D001122:Arginine Kinase