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

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• 作者：Susiji Wickramasinghe¹ ; ^{susijij@pdn.ac.lk" class="auth_mail" title="E-mail the corresponding author} ; ^{susijijp@yahoo.co.jp" class="auth_mail" title="E-mail the corresponding author} ; Lalani Yatawara² ; Mitsuru Nagataki³ ; Takeshi Agatsuma³
• 关键词：Toxocara canis ; Arginine kinase ; Gene structure ; Site directed mutagenesis ; Inhibition kinetics
• 刊名：Asian Pacific Journal of Tropical Medicine
• 出版年：2016
• 出版时间：October 2016
• 年：2016
• 卷：9
• 期：10
• 页码：995-1001
• 全文大小：1053 K

文摘

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.

Methods

Amplification of genomic DNA fragments containing introns was carried out by PCRs. The open-reading frame (1 200 bp) 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).

Results

Arginine kinase in T. canis has a seven-exon/six-intron gene structure. The lengths of the introns ranged from 542 bp to 2 500 bp. All introns begin with gt and end with ag. Furthermore, we measured the enzyme activity of site-directed mutants of the recombinant TCAK. The K_m value of the mutant (Alanine to Serine) decreased indicating a higher affinity for substrate arginine than the wild-type. The K_m value of the mutant (Serine to Glycine) increased to 0.19 mM. The K_m value (0.19 mM) of the double mutant (Alanine–Serine to Serine–Glycine) was slightly greater than in the wild-type (0.12 mM). 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.

Conclusions

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.