Catalytic Mechanism of RNA Backbone Cleavage by Ribonuclease H from Quantum Mechanics/Molecular Mechanics Simulations

Rosta, Edina; Nowotny, Marcin; Yang, Wei; Hummer, Gerhard

doi:10.1021/ja200173a

Cited by 181 publications

(330 citation statements)

References 46 publications

Supporting

Mentioning

309

Contrasting

Order By: Relevance

“…The observed concerted coupled proton transfer appears to be a general mechanism in enzymes that catalyzes phosphate transfer and cleavage reactions. 43,76,[95][96][97][98] Our calculations also provided evidence that the specific metal ion coordination mode may play a prominent role in the catalytic reaction. We developed a new implementation to quantitatively determine the symmetry around the metal ion during the catalytic reaction.…”

Section: Discussionmentioning

confidence: 53%

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

et al. 2015

Self Cite

View full text Add to dashboard Cite

Most enzymes present a catalytic mechanism where one or more proton transfer events occur coupled tightly together with the enzymatic chemical reaction. We show here that inactivating mutations decouple this proton transfer step from the phosphate cleavage reaction in dUTPase. Homotrimeric dUTPase enzymes catalyze the hydrolysis of dUTP to dUMP and pyrophosphate, using largely similar structural and functional groups as most AAA+ enzymes. dUTPases typically use a single Mg 2+ ion as a cofactor in the active site that is formed by direct protein-protein contacts including all three protomers. Here we focus on the C-terminal arm structural motif, which has sequence and functional similarities to P-loop motifs, and is required for catalysis. In this work, we have studied the functional roles of the C-terminal arm in ligand binding and catalysis by using QM/MM (quantum mechanics/molecular mechanics) calculations in conjunction with site-directed mutagenesis experiments. We also present a new method to assess the metal ion coordination symmetry during the catalytic reaction. Using this new implementation, we identified that the coordination symmetry follows a consistent pattern in the three systems studied, reaching the most symmetrical state near the transition states. We found that the phosphate cleavage proceeds with a concerted bimolecular (A N D N ) mechanism with a loose dissociative transition state, and that it is coupled with a proton transfer step involving a unanimously conserved Asp residue. We show that the main mechanistic effect of the lack of the C-terminal arm is to decouple the phosphate cleavage from the subsequent proton transfer step, resulting in a highbarrier altered reaction pathway.

show abstract

Section: Discussionmentioning

confidence: 53%

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

et al. 2015

Self Cite

View full text Add to dashboard Cite

show abstract

“…Following Ref. 39, we first determine the free energies F i of each N -replica simulation i (i = 1, . .…”

Section: Bioen Methods Combining Replica Simulations With Erosmentioning

confidence: 99%

Bayesian ensemble refinement by replica simulations and reweighting

Hummer

Köfinger

2015

The Journal of Chemical Physics

Self Cite

225

394

View full text Add to dashboard Cite

We describe different Bayesian ensemble refinement methods, examine their interrelation, and discuss their practical application. With ensemble refinement, the properties of dynamic and partially disordered (bio)molecular structures can be characterized by integrating a wide range of experimental data, including measurements of ensemble-averaged observables. We start from a Bayesian formulation in which the posterior is a functional that ranks different configuration space distributions. By maximizing this posterior, we derive an optimal Bayesian ensemble distribution. For discrete configurations, this optimal distribution is identical to that obtained by the maximum entropy "ensemble refinement of SAXS" (EROS) formulation.Bayesian replica ensemble refinement enhances the sampling of relevant configurations by imposing restraints on averages of observables in coupled replica molecular dynamics simulations. We show that the strength of the restraint should scale linearly with the number of replicas to ensure convergence to the optimal Bayesian result in the limit of infinitely many replicas. In the "Bayesian inference of ensembles" (BioEn) method, we combine the replica and EROS approaches to accelerate the convergence. An adaptive algorithm can be used to sample directly from the optimal ensemble, without replicas. We discuss the incorporation of singlemolecule measurements and dynamic observables such as relaxation parameters. The theoretical analysis of different Bayesian ensemble refinement approaches provides a basis for practical applications and a starting point for further investigations.

show abstract

“…This type of active site was first structurally investigated and a chemical mechanism proposed in the context of the 3′-5′ exonuclease of the Klenow fragment of E. coli DNA polymerase I (12). More recently, the mechanism has been further investigated and described for RNase H from Bacillus halodurans (13,14) and analyzed computationally with quantum mechanics/molecular mechanics (QM/MM) methods (15,16).…”

Section: Dna Transposases With Rnase H-like Catalytic Domainsmentioning

confidence: 99%

Mechanisms of DNA Transposition

Hickman

Dyda

2015

Microbiol Spectr

View full text Add to dashboard Cite

DNA transposases use a limited repertoire of structurally and mechanistically distinct nuclease domains to catalyze the DNA strand breaking and rejoining reactions that comprise DNA transposition. Here, we review the mechanisms of the four known types of transposition reactions catalyzed by (1) RNase H-like transposases (also known as DD(E/D) enzymes); (2) HUH single-stranded DNA transposases; (3) serine transposases; and (4) tyrosine transposases. The large body of accumulated biochemical and structural data, particularly for the RNase H-like transposases, has revealed not only the distinguishing features of each transposon family, but also some emerging themes that appear conserved across all families. The more-recently characterized single-stranded DNA transposases provide insight into how an ancient HUH domain fold has been adapted for transposition to accomplish excision and then site-specific integration. The serine and tyrosine transposases are structurally and mechanistically related to their cousins, the serine and tyrosine site-specific recombinases, but have to date been less intensively studied. These types of enzymes are particularly intriguing as in the context of site-specific recombination they require strict homology between recombining sites, yet for transposition can catalyze the joining of transposon ends to form an excised circle and then integration into a genomic site with much relaxed sequence specificity.In this chapter, we provide an overview of the fundamental concepts of DNA transposition mechanisms. Our aim is to emphasize basic themes and, in this effort, we will focus on specific illustrative cases rather than attempt an exhaustive review of the literature. We hope that the selected references will point the curious reader towards the landmark studies in the field as well as some of the most exciting recent results. We also direct the reader to other recent reviews (1-3).DNA transposases are enyzmes that move discrete segments of DNA called transposons from one location in the genome (often called the donor site) to a new site without using RNA intermediates. DNA transposases are usually encoded by the mobile element itself (in which case they are "autonomous" transposons). However, some transposons are missing a self-encoded transposase yet have ends that can be recognized by a transposase encoded somewhere else in the genome, and thus are "non-autonomous" (Siguier et al., this volume). Although logic suggests that all DNA transposons are moved by transposases, the term was originally reserved for those enzymes that do not require significant regions of homology between any part of the transposon and the sites to which they are moved, the so-called target (or insertion or integration) sites. As biology is not always neat and tidy, transposases can exhibit a spectrum of homology requirements and vagaries of terminology have arisen such that certain transposases are sometimes referred to as "resolvases" or by the generic term "recombinases".From a mechanistic perspective, t...

show abstract

Catalytic Mechanism of RNA Backbone Cleavage by Ribonuclease H from Quantum Mechanics/Molecular Mechanics Simulations

Cited by 181 publications

References 46 publications

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

Bayesian ensemble refinement by replica simulations and reweighting

Mechanisms of DNA Transposition

Contact Info

Product

Resources

About