Arginine kinase in Toxocara canis: Exon-intron organization, functional analysis of site-directed mutants and evaluation of putative enzyme inhibitors.

Abstract

To determine exon/intron organization of the Toxocara canis (T.canis) AK (TCAK) and to test green and black tea and several other chemicals against the activity of recombinant TCAK in the guanidino-specific region by site-directed mutants. Amplification of genomic DNA fragments containing introns was carried out by PCRs. The open-reading frame (1200bp) of TCAK (wild type) was cloned into the BamH1/SalI site of pMAL-c2X. The maltose-binding protein-TCAK fusion protein was expressed in Escherichia coli TB1 cells. The purity of the expressed enzyme was verified by SDS-PAGE. Mutations were introduced into the guanidino-specific region and other areas of pMAL/TCAK by PCR. Enzyme activity was measured with an NADH-linked assay at 25°C for the forward reaction (phosphagen synthesis). Arginine kinase in T.canis has a seven-exon/six-intron gene structure. The lengths of the introns ranged from 542bp to 2 500bp. All introns begin with gt and end with ag. Furthermore, we measured the enzyme activity of site-directed mutants of the recombinant TCAK. The Km value of the mutant (Alanine to Serine) decreased indicating a higher affinity for substrate arginine than the wild-type. The Km value of the mutant (Serine to Glycine) increased to 0.19mM. The Km value (0.19mM) of the double mutant (Alanine-Serine to Serine-Glycine) was slightly greater than in the wild-type (0.12mM). In addition, several other chemicals were tested; including plant extract Azadiracta indica (A.indica), an aminoglycoside antibiotic (aminosidine), a citrus flavonoid glycoside (rutin) and a commercially available catechin mixture against TCAK. Green and black tea (1:10 dilution) produced 15% and 25% inhibition of TCAK, respectively. The extract of A.indica produced 5% inhibition of TCAK. Moreover, green and black tea produced a non-competitive type of inhibition and A.indica produced a mixed-type of inhibition on TCAK. Arginine kinase in T.canis has a seven-exon/six-intron gene structure. However, further studies are needed to identify a specific compound within the extract causing the inhibitory effect and also to determine the molecular mechanisms behind inhibition of arginine kinase in T.canis.