title: Enzyme-promoted base flipping controls DNA methylation fidelity.
abstract: A quantitative understanding of how conformational transitions contribute to enzyme catalysis and specificity remains a fundamental challenge. A suite of biophysical approaches was used to reveal several transient states of the enzyme-substrate complexes of the model DNA cytosine methyltransferase M.HhaI. Multidimensional, transverse relaxation-optimized nuclear magnetic resonance (NMR) experiments show that M.HhaI has the same conformation with noncognate and cognate DNA sequences. The high-affinity cognatelike mode requires the formation of a subset of protein-DNA interactions that drive the flipping of the target base from the helix to the active site. Noncognate substrates lacking these interactions undergo slow base flipping, and fluorescence tracking of the catalytic loop corroborates the NMR evidence of a loose, nonspecific binding mode prior to base flipping and subsequent closure of the catalytic loop. This slow flipping transition defines the rate-limiting step for the methylation of noncognate sequences. Additionally, we present spectroscopic evidence of an intermediate along the base flipping pathway that has been predicted but never previously observed. These findings provide important details of how conformational rearrangements are used to balance specificity with catalytic efficiency.
authors: Department of Chemistry and Biochemistry, University of California , Santa Barbara, California 93106, United States.
authors: Douglas M
authors: DM
authors: Matje
authors: Hongjun
authors: H
authors: Zhou
authors: Darren A
authors: DA
authors: Smith
authors: Robert K
authors: RK
authors: Neely
authors: David T F
authors: DT
authors: Dryden
authors: Anita C
authors: AC
authors: Jones
authors: Frederick W
authors: FW
authors: Dahlquist
authors: Norbert O
authors: NO
authors: Reich
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D020134:Catalytic Domain / Q000235:genetics; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D019175:DNA Methylation / Q000502:physiology*; D015257:DNA-Cytosine Methylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004247:DNA; C037664:DNA modification methylase HhaI; D015257:DNA-Cytosine Methylases