Characterization of the large subunit of EcoHK31I methyltransferase by structural modeling and mutagenesis
-
Amanda N.-S. Mak
Abstract
M.EcoHK31I is a naturally occurring mC5-methyltransferase with a large α polypeptide and a small β polypeptide. Polypeptide α contains conserved motifs I–VIII and X, and polypeptide β contains motif IX. To understand how polypeptide α carries out its function, a molecular model of the large domain of polypeptide α was generated using M.HhaI and M.HaeIII as templates. The large domain is a mixed α/β structure. Residues 15–19 in motif I (Phe-Naa-Gly-Naa) are conserved for cofactor binding. The key catalytic residue Cys-79 in motif IV is also conserved in comparison with other C-5 MTases. Comparing polypeptide α with M.HhaI and M.HaeIII revealed a unique region upstream of motif X. To understand the role of this region, 14 charged residues between R224 and E271 in the putative small domain were mutated. Activity assays indicated that most of these charges can be eliminated or changed conservatively. Among these charged residues, R224, E240, D245 and D251 may take part in proper interaction with DNA in the presence of polypeptide β.
References
Bhattacharya, S.K. and Dubey, A.K. (2002). The N-terminus of m5C-DNA methyltransferase MspI is involved in its topo- isomerase activity. Eur. J. Biochem.269, 2491–2497.10.1046/j.1432-1033.2002.02913.xSuche in Google Scholar
Cesnaviciene, E., Petrusyte, M., Kazlauskiene, R., Maneliene, Z., Timinskas, A., Lubys, A., and Janulaitis, A. (2001). Characterization of AloI, a restriction-modification system of a new type. J. Mol. Biol.314, 205–216.10.1006/jmbi.2001.5049Suche in Google Scholar
Cheng, X., Kumar, S., Posfai, J., Pflugrath, J.W., and Roberts, R.J. (1993). Crystal structure of the HhaI DNA methyltransferase complexed with S-adenosyl-l-methionine. Cell74, 299–307.10.1016/0092-8674(93)90421-LSuche in Google Scholar
Dryden, D.T.F. (1999). Bacterial DNA methyltransferases. In: S- Adenosylmethionine-Dependent Methyltransferases: Structures and Functions, X. Cheng and R.M. Blumenthal, eds. (Singapore: World Scientific), pp. 283–340.10.1142/9789812813077_0011Suche in Google Scholar
Fung, W.T., Sze, K.H., Lee, K.F., and Shaw, P.C. (2006). Functional studies of the small subunit of EcoHK31I DNA methyltransferase. Biol. Chem.387, 507–513.10.1515/BC.2006.066Suche in Google Scholar
Heithoff, D.M., Sinsheimer, R.L., Low, D.A., and Mahan, M.J. (1999). An essential role for DNA adenine methylation in bacterial virulence. Science284, 967–970.10.1126/science.284.5416.967Suche in Google Scholar
Karreman, C. and de Waard, A. (1990). Agmenellum quadruplicatum M.AquI, a novel modification methylase. J. Bacteriol.172, 266–272.Suche in Google Scholar
Klimasauskas, S., Kumar, S., Roberts, R.J., and Cheng, X. (1994). HhaI methyltransferase flips its target base out of the DNA helix. Cell76, 357–369.10.1016/0092-8674(94)90342-5Suche in Google Scholar
Kobayashi, I. (2001). Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution. Nucleic Acids Res.29, 3742–3756.10.1093/nar/29.18.3742Suche in Google Scholar PubMed PubMed Central
Laskowski, R.A., Moss, D.S., and Thornton, J.M. (1993). Main- chain bond lengths and bond angles in protein structures. J. Mol. Biol.231, 1049–1067.10.1006/jmbi.1993.1351Suche in Google Scholar PubMed
Lee, K.F., Kam, K.M., and Shaw, P.C. (1995). A bacterial methyltransferase M.EcoHK311 requires two proteins for in vitro methylation. Nucleic Acids Res. 23, 103–108.10.1093/nar/23.1.103Suche in Google Scholar PubMed PubMed Central
Lee, K.F., Liaw, Y.C., and Shaw, P.C. (1996). Overproduction, purification and characterization of M.EcoHK31I, a bacterial methyltransferase with two polypeptides. Biochem. J.314, 321–326.Suche in Google Scholar
Lindstrom, W.M. Jr., Malygin, E.G., Ovechkina, L.G., Zinoviev, V.V., and Reich, N.O. (2003). Functional analysis of BamHI DNA cytosine-N4 methyltransferase. J. Mol. Biol.325, 711–720.10.1016/S0022-2836(02)01282-2Suche in Google Scholar
McNamara, A.R., Hurd, P.J., Smith, A.E., and Ford, K.G. (2002). Characterisation of site-biased DNA methyltransferases: specificity, affinity and subsite relationships. Nucleic Acids Res.30, 3818–3830.10.1093/nar/gkf501Suche in Google Scholar
Naito, T., Kusano, K., and Kobayashi, I. (1995). Selfish behavior of restriction-modification systems. Science267, 897–899.10.1126/science.7846533Suche in Google Scholar
Pinarbasi, H., Pinarbasi, E., and Hornby, D.P. (2003). The small subunit of M. AquI is responsible for sequence-specific DNA recognition and binding in the absence of the catalytic domain. J. Bacteriol.185, 1284–1288.Suche in Google Scholar
Reinisch, K.M., Chen, L., Verdine, G.L., and Lipscomb, W.N. (1995). The crystal structure of HaeIII methyltransferase convalently complexed to DNA: an extrahelical cytosine and rearranged base pairing. Cell82, 143–153.10.1016/0092-8674(95)90060-8Suche in Google Scholar
Rost, B., Yachdav, G., and Liu, J. (2004). The PredictProtein server. Nucleic Acids Res.32, 321–326.10.1093/nar/gkh377Suche in Google Scholar
Sambrook, J. and Russell, D.W. (2001). Molecular Cloning: A Laboratory Manual, Vol. 2, 3rd edition (Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory Press), pp. 9.1–10.48.Suche in Google Scholar
Sankpal, U.T. and Rao, D.N. (2002a). Structure, function, and mechanism of HhaI DNA methyltransferases. Crit. Rev. Biochem. Mol. Biol.37, 167–197.10.1080/10409230290771492Suche in Google Scholar
Sankpal U.T. and Rao, D.N. (2002b). Mutational analysis of conserved residues in HhaI DNA methyltransferase. Nucleic Acids Res.30, 2628–2638.10.1093/nar/gkf380Suche in Google Scholar
Sethmann, S., Ceglowski, P., Willert, J., Iwanicka-Nowicka, R., Trautner, T.A., and Walter, J. (1999). M.(ϕ)BssHII, a novel cytosine-C5-DNA-methyltransferase with target-recognizing domains at separated locations of the enzyme. EMBO J.18, 3502–3508.Suche in Google Scholar
Shenkin, P.S., Yarmush, D.L., Fine, R.M., Wang, H.J., and Levinthal, C. (1987). Predicting antibody hypervariable loop conformation. I. Ensembles of random conformations for ringlike structures. Biopolymers26, 2053–2085.Suche in Google Scholar
Zakharova, M.V., Beletskaya, I.V., Kravetz, A.N., Pertzev, A.V., Mayorov, S.G., Shlyapnikov, M.G., and Solonin, A.S. (1998). Cloning and sequence analysis of the plasmid-borne genes encoding the Eco29kI restriction and modification enzymes. Gene208, 177–182.10.1016/S0378-1119(97)00637-9Suche in Google Scholar
Zhang, X., Zhou, L., and Cheng, X. (2000). Crystal structure of the conserved core of protein arginine methyltransferase PRMT3. EMBO J.19, 3509–3519.10.1093/emboj/19.14.3509Suche in Google Scholar PubMed PubMed Central
©2007 by Walter de Gruyter Berlin New York
Artikel in diesem Heft
- Supplementary material to the paper “Evolutionary selection pressure and family relationships among connexin genes”
- Evolutionary selection pressure and family relationships among connexin genes
- Characterization of the large subunit of EcoHK31I methyltransferase by structural modeling and mutagenesis
- Purification, characterization, and molecular gene cloning of an antifungal protein from Ginkgo biloba seeds
- Maximal Ca2+i stimulation of cardiac Na+/Ca2+ exchange requires simultaneous alkalinization and binding of PtdIns-4,5-P2 to the exchanger
- A highly conserved protein secreted by the prostate cancer cell line PC-3 is expressed in benign and malignant prostate tissue
- Properties and partial purification of sialate-O-acetyltransferase from bovine submandibular glands
- Raft association and lipid droplet targeting of flotillins are independent of caveolin
- On the presence of C2-ceramide in mammalian tissues: possible relationship to etherphospholipids and phosphorylation by ceramide kinase
- Specific inhibition of interleukin-13 activity by a recombinant human single-chain immunoglobulin domain directed against the IL-13 receptor α1 chain
- Effects of disease-modifying anti-rheumatic drugs (DMARDs) on the activities of rheumatoid arthritis-associated cathepsins K and S
- Compartmentalised expression of meprin in small intestinal mucosa: enhanced expression in lamina propria in coeliac disease
- Human dipeptidyl peptidase III acts as a post-proline-cleaving enzyme on endomorphins
- Transgenic mouse brains for the evaluation and quality control of BSE tests
Artikel in diesem Heft
- Supplementary material to the paper “Evolutionary selection pressure and family relationships among connexin genes”
- Evolutionary selection pressure and family relationships among connexin genes
- Characterization of the large subunit of EcoHK31I methyltransferase by structural modeling and mutagenesis
- Purification, characterization, and molecular gene cloning of an antifungal protein from Ginkgo biloba seeds
- Maximal Ca2+i stimulation of cardiac Na+/Ca2+ exchange requires simultaneous alkalinization and binding of PtdIns-4,5-P2 to the exchanger
- A highly conserved protein secreted by the prostate cancer cell line PC-3 is expressed in benign and malignant prostate tissue
- Properties and partial purification of sialate-O-acetyltransferase from bovine submandibular glands
- Raft association and lipid droplet targeting of flotillins are independent of caveolin
- On the presence of C2-ceramide in mammalian tissues: possible relationship to etherphospholipids and phosphorylation by ceramide kinase
- Specific inhibition of interleukin-13 activity by a recombinant human single-chain immunoglobulin domain directed against the IL-13 receptor α1 chain
- Effects of disease-modifying anti-rheumatic drugs (DMARDs) on the activities of rheumatoid arthritis-associated cathepsins K and S
- Compartmentalised expression of meprin in small intestinal mucosa: enhanced expression in lamina propria in coeliac disease
- Human dipeptidyl peptidase III acts as a post-proline-cleaving enzyme on endomorphins
- Transgenic mouse brains for the evaluation and quality control of BSE tests