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 Taxocara 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 isoclectric 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 mu mol Pi/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 (248.19 s(-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. (c) 2007 Published by Elsevier Inc.