Mechanism-Based Inhibition of an Essential Bacterial Adenine DNA Methyltransferase:  Rationally Designed Antibiotics

Wahnon, Daphne; Shier, Vincent K.; Benkovic, Stephen J.

doi:10.1021/ja003285o

Cited by 32 publications

(32 citation statements)

References 19 publications

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“…For example, the extensive medicinal chemistry efforts to develop AdoMet antagonists have met with limited success, presumably in part due to the interference with the plethora of AdoMet-dependent enzymes. [15][16][17] Although strategies to develop drugs based on compounds that interfere with AdoMet binding can be exploited, 18 such drugs are likely to reveal specificity and toxicity profiles distinct from compounds that act, for example, by interfering with DNA binding.…”

Section: Discussionmentioning

confidence: 99%

Selective Inhibitors of Bacterial DNA Adenine Methyltransferases

Mashhoon

Pruss²,

Carroll³

et al. 2006

SLAS Discovery

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Section: Discussionmentioning

confidence: 99%

Selective Inhibitors of Bacterial DNA Adenine Methyltransferases

Mashhoon

Pruss²,

Carroll³

et al. 2006

SLAS Discovery

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“…These enzymes decorate the DNA major groove with a methyl group at cytosine (C 5 or N 4 ) or adenine (N 6 ), thereby providing an epigenetic signature with diverse biological consequences. The bacterial and human enzymes are the targets of novel antibiotics (18) and cancer drug development efforts (19), respectively. M.HhaI is representative of many DNA methyltransferases because it retains highly conserved motifs associated with substrate recognition and catalysis (20).…”

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confidence: 99%

Statistical coevolution analysis and molecular dynamics: Identification of amino acid pairs essential for catalysis

Estabrook

Luo

Purdy

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Molecular dynamics (MD) simulations of HhaI DNA methyltransferase and statistical coupling analysis (SCA) data on the DNA cytosine methyltransferase family were combined to identify residues that are coupled by coevolution and motion. The highest ranking correlated pairs from the data matrix product (SCA⅐MD) are colocalized and form stabilizing interactions; the anticorrelated pairs are separated on average by 30 Å and form a clear focal point centered near the active site. We suggest that these distal anticorrelated pairs are involved in mediating active-site compressions that may be important for catalysis. Mutants that disrupt the implicated interactions support the validity of our combined SCA⅐MD approach.anticorrelated motion ͉ correlated motion ͉ M.HhaI ͉ statistical coupling analysis T he proposal that protein dynamics contributes significantly to enzyme catalysis is intriguing (1-4) yet is supported by limited experimental evidence. Previous studies have shown that correlated and anticorrelated motions within an enzyme's active site enhance the reaction rate by various mechanisms that increase the relative amounts of reactive orientations (5). These active-site fluctuations are proposed to result from motions involving distal structural elements and interconnecting networks (1-4). This hypothesis is indirectly supported by emerging molecular dynamics (MD) (1, 2), NMR (6, 7), and hybrid approaches (8-12). The MD studies, although difficult to verify experimentally, have provided highly suggestive results relating dynamics to catalysis. Ultimately, the quantitative contribution to catalysis of various dynamic mechanisms requires direct experimental testing. We combined MD simulations and a coevolution analysis [statistical coupling analysis (SCA); ref. 13] to identify residues that are coupled by coevolution and motion.Although MD simulations reveal active-site correlated and anticorrelated motions, the identity and role of specific structural elements outside the active site in mediating such motions is difficult to assign. For example, MD cross-correlation analyses are dominated by anticorrelated motions occurring between the most distal regions of protein, often residing in distinct domains (5). Although MD simulations implicate regions of allowed motion, the identity of single amino acids that facilitate these motions is not forthcoming and hence difficult for protein engineers to test. SCA identifies the functional coupling of specific residue pairs that in many cases are distal in the three-dimensional structure. The coupling of such residues leads to their coevolution and is revealed by the statistical analysis of hundreds of related sequences; this approach recently was validated by NMR and protein engineering studies (13-16). These applications of SCA have been focused on protein-ligand interactions, and here we apply SCA toward protein dynamics and catalysis.M.HhaI is one of many S-adenosylmethionine (AdoMet)-dependent DNA-modifying enzymes found in bacteria, plants, and animals (17). These enzym...

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“…The enzymes are structurally characterized, carry out both DNA bending and base flipping (8), and the bacterial and human enzymes are the targets of novel antibiotic (9) and cancer drug development efforts respectively (10). These largely monomeric, AdoMet-dependent enzymes modify adenine (N 6 ) or cytosine (N 4 or C 5 ) within duplex DNA by delivering a methyl moiety into the major groove.…”

mentioning

confidence: 99%

Simultaneous DNA Binding, Bending, and Base Flipping

Hopkins

Reich

2004

Journal of Biological Chemistry

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We measured the kinetics of DNA bending by M.EcoRI using DNA labeled at both 5-ends and observed changes in fluorescence resonance energy transfer. Although known to bend its cognate DNA site, energy transfer is decreased upon enzyme binding. This unanticipated effect is shown to be robust because we observe the identical decrease with different dye pairs, when the dye pairs are placed on the respective 3-ends, the effect is cofactor-and protein-dependent, and the effect is observed with duplexes ranging from 14 through 17 base pairs. The same labeled DNA shows the anticipated increased energy transfer with EcoRV endonuclease, which also bends this sequence, and no change in energy transfer with EcoRI endonuclease, which leaves this sequence unbent. We interpret these results as evidence for an increased end-to-end distance resulting from M.EcoRI binding, mediated by a mechanism novel for DNA methyltransferases, combining DNA bending and an overall expansion of the DNA duplex. The M.EcoRI protein sequence is poorly accommodated into well defined classes of DNA methyltransferases, both at the level of individual motifs and overall alignment. Interestingly, M.EcoRI has an intercalation motif observed in the FPG DNA glycosylase family of repair enzymes. Enzyme-dependent changes in anisotropy and fluorescence resonance energy transfer have similar rate constants, which are similar to the previously determined rate constant for base flipping; thus, the three processes are nearly coincidental. Similar fluorescence resonance energy transfer experiments following AdoMet-dependent catalysis show that the unbending transition determines the steady state product release kinetics.Structural transitions involving substrate-induced changes in enzyme conformation and protein-induced changes in substrate conformation occur in diverse classes of enzymes. The quantitative importance of such mechanisms toward catalysis and specificity has been demonstrated for DNA polymerases (1), phytase (2), integration host factor (3), pyridoxal kinase (4), restriction enzymes, DNA repair enzymes (5), and DNA modifying enzymes (6). Yet, conformational mechanisms are difficult to study because the reaction intermediates are largely inaccessible to classical kinetic analysis. Spectroscopic probes and, in particular, the highly distant-dependent method of fluorescence resonance energy transfer (FRET) 1 provide a viable solution-based approach to characterize conformational intermediates (7). Only through the quantitative analysis of stable conformational intermediates and the kinetic transitions involving their interconversion can a mechanistic understanding of catalysis and specificity be approached.DNA methyltransferases provide a rich system to investigate the mechanisms and importance of conformational transitions. The enzymes are structurally characterized, carry out both DNA bending and base flipping (8), and the bacterial and human enzymes are the targets of novel antibiotic (9) and cancer drug development efforts respectively (10). These l...

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Mechanism-Based Inhibition of an Essential Bacterial Adenine DNA Methyltransferase: Rationally Designed Antibiotics

Cited by 32 publications

References 19 publications

Selective Inhibitors of Bacterial DNA Adenine Methyltransferases

Selective Inhibitors of Bacterial DNA Adenine Methyltransferases

Statistical coevolution analysis and molecular dynamics: Identification of amino acid pairs essential for catalysis

Simultaneous DNA Binding, Bending, and Base Flipping

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