| Summary: | 碩士 === 國立中興大學 === 生物科技學研究所 === 93 === Bamboo mosaic virus ( BaMV ), a potexvirus in alphavirus-like superfamily, is a positive-stranded RNA virus with five open reading frames ( ORFs ). The 155-kDa polypeptide, encoded by ORF1 of BaMV, consists of a AdoMet-dependent guanylyltransferase domain ( capping enzyme ), an RNA helicase-like domain, and an RNA-dependent RNA polymerase ( RdRp ) domain, in order of from N to C termini. Previous studies of the BaMV capping enzyme has been shown that the substitution of conservative H68 to alanine could increase the methyltransferase activity, but completely abolished the guanylyltransferase activity. The first part of this study is to characterize the mutant capping enzyme H68A. Besides using GTP, the wild type capping enzyme could also forms the intermediate of m7GMP-enzyme complex by using GDP as the initial substrate; however the H68A mutant only forms m7GDP by using GDP. When adding RNA, the H68A mutant doses not change the methyltransferase activity. Furthermore, we analyzed the KM values of H68A mutant enzyme for AdoMet and GTP, which were 26μM and 65μM, respectively. The second part, to understand the roles of aromatic amino acid residues are responsible for the methyltransferase activity of BaMV capping enzyme. Based on H68A mutant generates Twenty-one of aromatic amino acid residues substitutional double mutations by site-directed mutagenesis. These results shown that H68A/F161A, H68A/Y192A, H68A/Y192F, H68A/Y203F, H68A/Y213A, H68A/Y213F, H68A/W222A, H68A/Y340A, H68A/W311A, H68A/W311F, H68A/Y338A, H68A/Y338F are severely decreased methyltransferase activity than H68A mutant. Moreover, H68A/Y126A, H68A/W296A, H68A/F144A and H68A/F384A could shift the enzymatic specific form GTP to GDP, and those mutants H68A/Y126A, H68A/F144A, H68A/Y203A, H68A/W296A, H68A/F339A and H68A/F384A have increased the substrate affinity of AdoMet on the methyltransferase activity.
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