“…Furthermore, this loss of DNA affinity can be accomplished by perturbing interactions with the base of the target nucleotide, even though the WT enzyme is highly accommodating for mismatched nucleotides. One obvious interaction previously suggested to contribute to the base flipping process involves protein-phosphate interactions (8,27,31,(43)(44)(45); for M.HhaI, this includes the phosphate 5Ј to the flipped cytosine and Arg 165 . Below we discuss this in detail in the context of a conserved motif, but we propose that any perturbation in this interaction is unlikely to contribute significantly to the mutant's loss in cognate DNA binding affinity.…”
The molecular basis of enzymatic catalysis is of broad interest, with implications for biocatalyst design and drug development. The abundance of detailed three-dimensional structures and investigational methods provides newly addressable aspects of enzymatic function. We are interested in the importance of protein motion, and particularly correlated motions, to catalysis. The underlying premise is that protein-solvent interactions are converted into peptide motions, resulting in the transient stabilization of active site elements with preferred reactivities (1, 2).Recent studies (1) have provided highly suggestive evidence for this concept. Molecular dynamics investigations of dihydrofolate reductase demonstrate that strong coupled motions in the reactive complex disappear in the product complexes, indicating that these motions may be linked to catalysis. Mutants that alter the kinetics of particular catalytic steps are concentrated within segments of the protein structure shown to participate in highly correlated motions (1). Solid state NMR and solution NMR relaxation studies have measured substrate and protein dynamics that are matched to the turnover time of the respective enzymes (3). Studies of hydrogen and electron tunneling during enzyme catalysis provide further evidence for the importance of protein dynamics to catalytic events at the active site (4, 5).Molecular dynamic simulations of catechol O-methyltransferase and M.HhaI 1 DNA methyltransferase provided initial evidence for correlated motions within the active sites of these enzymes (6, 7). We sought to test the importance to catalysis of motions made by specific distal residues (His 127 -Thr 132 ) in facilitating active site chemistries by altering the position and orientation of critical residues such as Val 121 . Alanine scan point mutagenesis and kinetic characterization of individual steps in the catalytic cycle were used to probe the effects of such mutations and provide insights into the roles of correlated motions. M.HhaI provides an excellent structurally and functionally tractable enzyme to study various aspects of catalysis, including base flipping and the importance of motions to catalysis. M.HhaI, from Haemophilus haemolyticus, is an AdoMetdependent C 5 -cytosine methyltransferase that methylates the central cytosine (C) in the recognition sequence 5Ј-GCGC-3Ј after stabilizing the target base in an extrahelical position. Many M.HhaI crystal structures provide structural insights into the mechanisms of DNA methylation and base flipping (8). Functional analysis of the WT M.HhaI has been extensive (9 -12), including K D DNA determination for a variety of DNA substrates (13,14). Many structural components of the M.HhaI mechanism have been examined by mutagenesis including Gln 237 , which positions itself into the DNA helix and interacts with the lone guanine (15), and Cys 81 , which forms a covalent bond to the target cytosine (16). Other mutational studies have examined protein-phosphate interactions (17) and conserved residues within the...
“…These lie below the plane of the base and likely induce the nitrogen to switch from the planar sp 2 orbital configuration it normally possesses, to the tetrahedral sp 3 configuration (105). In this latter configuration, the lone pair orbital of nitrogen (purple stick) is appropriately positioned for in-line nucleophilic attack on the carbon thiol (pink) of SAM, initiating the DNA methylation reaction (105,106). …”
Section: Type I Restriction Enzymesmentioning
confidence: 99%
“…The four catalytic aa are located in a slot in the surface of the protein into which the target base flips before methyl transfer. The aromatic aa stacks with the flipped base, compensating for the loss of DNA base stacking (210), and the exocyclic amino group forms two hydrogen bonds with the protein, compensating for the loss of the Watson–Crick base-pairing hydrogen bonds (105,106) (Figure 2). Methyl transfer occurs directly, without formation of a covalent protein–DNA intermediate such as occurs in the m5C-MTases.…”
Type I restriction enzymes (REases) are large pentameric proteins with separate restriction (R), methylation (M) and DNA sequence-recognition (S) subunits. They were the first REases to be discovered and purified, but unlike the enormously useful Type II REases, they have yet to find a place in the enzymatic toolbox of molecular biologists. Type I enzymes have been difficult to characterize, but this is changing as genome analysis reveals their genes, and methylome analysis reveals their recognition sequences. Several Type I REases have been studied in detail and what has been learned about them invites greater attention. In this article, we discuss aspects of the biochemistry, biology and regulation of Type I REases, and of the mechanisms that bacteriophages and plasmids have evolved to evade them. Type I REases have a remarkable ability to change sequence specificity by domain shuffling and rearrangements. We summarize the classic experiments and observations that led to this discovery, and we discuss how this ability depends on the modular organizations of the enzymes and of their S subunits. Finally, we describe examples of Type II restriction–modification systems that have features in common with Type I enzymes, with emphasis on the varied Type IIG enzymes.
“…17 Furthermore, a push mechanism was proposed in which M.TaqI -by domain closure -pushes actively against the target base partner thymine, which in turn displaces the target adenine from the inside of the DNA helix. 18 However, it is not always possible to obtain evidence of base flipping by using X-ray crystallography, as crystals are not always obtainable. Therefore, spectroscopic methods have often been adopted.…”
We report the crystal structure of the DNA adenine-N6 methyltransferase, M.TaqI, complexed with DNA, showing the fluorescent adenine analog, 2-aminopurine, flipped out of the DNA helix and occupying virtually the same position in the active site as the natural target adenine. Time-resolved fluorescence spectroscopy of the crystalline complex faithfully reports this state: base flipping is accompanied by the loss of the very short ( approximately 50 ps) lifetime component associated with fully base-stacked 2-aminopurine in DNA, and 2-aminopurine is subject to considerable quenching by pi-stacking interactions with Tyr108 in the catalytic motif IV (NPPY). This proves 2-aminopurine to be an excellent probe for studying base flipping by M.TaqI and suggests similar quenching in the active sites of DNA and RNA adenine-N6 as well as DNA cytosine-N4 methyltransferases sharing the conserved motif IV. In solution, the same distinctive fluorescence response confirms complete destacking from DNA and is also observed when the proposed key residue for base flipping by M.TaqI, the target base partner thymine, is substituted by an abasic site analog. The corresponding cocrystal structure shows 2-aminopurine in the active site of M.TaqI, demonstrating that the partner thymine is not essential for base flipping. However, in this structure, a shift of the 3' neighbor of the target base into the vacancy left after base flipping is observed, apparently replicating a stabilizing role of the missing partner thymine. Time-resolved fluorescence and acrylamide quenching measurements of M.TaqI complexes in solution provide evidence for an alternative binding site for the extra-helical target base within M.TaqI and suggest that the partner thymine assists in delivering the target base into the active site.
“…This type of structural distortion of the DNA enables the catalytic enzyme to access the specific base and perform chemical reactions on it. For example, M. Hha I catalyzes the transfer of methyl group from S-adenosyl- l -methionine (SAM) to the target base cytosine, the mechanism of which has been studied extensively (Figure ). − Over the years, numerous crystal structures of protein−DNA complexes where base flipping occurs have been reported, including several methyltransferases (M. Hha I, , M. Hae III, and M. Taq I , ), glycosylases , (T4 endonuclease V, human UDG, − Escherichia coli MUG, human AAG, E. coli AlkA, and bOGG1) and endonucleases ( E. coli endonuclease IV and HAP1). Clearly, base flipping, as it is commonly known, is a phenomenon important for the biological function of both DNA and RNA. ,,, 1 Crystal structure of the ternary complex of M. Hha I, DNA, and SAH (PDB ID: 1MHT) generated using VMD .…”
scite is a Brooklyn-based startup that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
