Toxocara canis: Molecular cloning, characterization, expression and comparison of the kinetics of cDNA-derived arginine kinase

Abstract

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 (p I) 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 μmol 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.