? The prokaryotic Hhal DMA methyltransferase (M.Hhal) is part of a larger class of enzymes, some of which are antibiotic or anticancer targets. The availability of M.Hhal crystal structures makes this form amenable for mechanistic studies. The proposed research will address the catalytic role of protein segments outside of the active site. The contribution of protein motion to the chemical steps of catalysis within an enzyme sub- strate complex is a subject of intense debate. Molecular dynamics simulations on M.Hhal indicate that some segments of the protein remote in sequence and structure move in concert. As some of these correlated motions traverse the active site, their significance to catalysis will be probed by random mutagenesis targeted to these segments coupled with a high throughput screen. Whether mutants affecting catalysis but removed from the active site do so through perturbations in the equilibrium structure or protein dynamics will be determined by computational and experimental means. Genetic co-variation will also serve as a method for suggesting important residues distant from the active site. Study of the effects of distal mutations in enzymes will aid in understanding genetic diseases, antibiotic resistance, and protein engineering. ? ?
| Purdy, Matthew M; Holz-Schietinger, Celeste; Reich, Norbert O (2010) Identification of a second DNA binding site in human DNA methyltransferase 3A by substrate inhibition and domain deletion. Arch Biochem Biophys 498:13-22 |
| Zhou, Hongjun; Purdy, Matthew M; Dahlquist, Frederick W et al. (2009) The recognition pathway for the DNA cytosine methyltransferase M.HhaI. Biochemistry 48:7807-16 |
