Determinants of sequence-specific DNA methylation: target recognition and catalysis are coupled in M.HhaI.
Sequence specificity studies of the wild-type bacterial DNA cytosine C5 methyltransferase HhaI were carried out with cognate (5'GCGC3') and noncognate DNA substrates containing single base pair changes at the first and the fourth position (underlined). Specificity for noncognate site methylation at the level of kcat/KDDNA is decreased 9000-80000-fold relative to the cognate site, manifested through changes in methylation, or a prior step, and changes in KDDNA. Analysis of a new high-resolution enzyme-DNA cocrystal structure provides a partial mechanistic understanding of this discrimination. To probe the significance of conformational transitions occurring prior to catalysis in determining specificity, we analyzed the double mutant (H127A/T132A). These amino acid substitutions disrupt the interface between the flexible loop (residues 80-99), which interacts with the DNA minor groove, and the active site. The mutant's methylation of the cognate site is essentially unchanged, yet its methylation of noncognate sites is decreased up to 460-fold relative to the wild-type enzyme. We suggest that a significant contribution to M.HhaI's specificity involves the stabilization of reaction intermediates prior to methyl transfer, mediated by DNA minor groove-protein flexible loop interactions.
Youngblood
Ben
B
Department of Chemistry and Biochemistry and Program in Biomolecular Science and Engineering, University of California, Santa Barbara, California 93106-9510.
Buller
Fabian
F
Reich
Norbert O
NO
D019943:Amino Acid Substitution / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D017124:Conserved Sequence / Q000235:genetics; D019175:DNA Methylation; D004252:DNA Mutational Analysis; D015257:DNA-Cytosine Methylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D013816:Thermodynamics
C037664:DNA modification methylase HhaI; D015257:DNA-Cytosine Methylases