Functional Analysis of Histidine 68 on the RNA Capping Enzyme of Bamboo Mosaic Virus

• Main Authors: Chiao-Yuan Yu, 游喬媛
• Other Authors: Meng-Menghsiao
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/68952927575524288456

碩士 === 國立中興大學 === 生物科技學研究所 === 92 === Open reading frame 1 (ORF1) of Bamboo mosaic virus (BaMV) encodes a 155-kDa replicase that contains capping enzyme domain, helicase-like domain, and RNA-dependent RNA polymerase (RdRp) domain, in order of from N to C termini. The viral capping enzyme domain expressed in Saccharomyces cerevisae exhibits both GTP methyltransferase (MTase) and S-adenosylmethionine (AdoMet) - dependent guanylyltransferase (GTase) activities. The MTase transfers the methyl group from AdoMet to GTP; subsequently, the GTase catalyzes transguanylation via the covalent m7GMP-enzyme intermediate complex. Substitution of the conserved H68 with alanine increased the MTase activity but completely abolished the GTase activity. Taken together with the fact that m7GMP links to the viral enzyme through phosphoramide bond, it has been suggested that the H68 residue might form the covalent bond to m7GMP during transguanylation. In order to understand the function of H68, the residue was replaced by Cys, Lys, Asn, Gln, Arg, Ser, Thr, or Tyr. We hope that some of the mutant enzymes will regain the GTase activity so that we are able to examine our hypothesis by looking over the acid or base stability of the m7GMP-enzyme intermediate. The various mutated proteins were expressed in yeast and purified by metal affinity chromatography in this study. The GTase activity, detected by the formation of 32P-labeled proteins after incubating the proteins with [α-32P] GTP in the present of AdoMet, was not detected in all mutant proteins. Meanwhile, the MTase activity of the mutated enzymes was determined by the transfer of 3H-methyl group from Ado[methyl-3H]Met to GTP. Mutant enzymes（H68C, H68K, and H68S）have the MTase activity increased, but other mutant proteins display lower MTase activity comparing with that of the wild-type enzyme. The results suggest that the imidazole side chain of H68 is not necessary for the MTase activity; however, local conformational changes induced by mutations may play a role for substrate binding. In this study, we are unable to determine that H68 is the residue links to the m7GMP. In the future, structural evidence such as peptide mapping is needed to define the residues for forming of the covalent bond to m7GMP.