Calcium Inhibition of Ribonuclease H1 Two-Metal Ion Catalysis

Rosta, Edina; Yang, Wei; Hummer, Gerhard

doi:10.1021/ja411408x

Cited by 56 publications

(76 citation statements)

References 93 publications

Supporting

Mentioning

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: 54%

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: 54%

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

“…1c). These differences explain why Ca 2+ inhibits RNase H1 16 and also the observation that sulfur replacement of the pro-Rp oxygen of the 3′-phosphate, which coordinates K + u , reduces the catalytic rate of E. coli RNase H1 by seven fold 17 . Despite binding of the two Mg 2+ and the presence of K + u , the nucleophilic water is misaligned by 20° and too distant (3.3 Å) for the in-line nucleophilic attack (Fig.…”

Section: Resultsmentioning

confidence: 99%

Cation trafficking propels RNA hydrolysis

Samara

Yang

2018

Nat Struct Mol Biol

Self Cite

124

View full text Add to dashboard Cite

Catalysis by the RNase H-superfamily members is generally believed to require only two Mg2+ ions coordinated by active-site carboxylates. By examining the catalytic process of B. Halodurans RNase H1 in crystallo, however, we find that the two canonical Mg2+ ions and an additional K+ fail to align the nucleophilic water for RNA cleavage. Substrate alignment and product formation require a second K+ and a third Mg2+, which replaces the first K+ and departs immediately after cleavage. A third transient Mg2+ has also been observed for DNA synthesis, but there it coordinates the leaving group instead of the nucleophile as in this hydrolysis reaction. These transient cations have no contact with enzymes. Other DNA and RNA enzymes that catalyze consecutive cleavage and strand transfer reactions in a single active site may likewise require cation trafficking coordinated by substrate.

show abstract

“…The only divalent metal ion species that appear to be able to support all the steps of transposition are Mg (22). However, it has been reported that Ca 2+ can catalyze strand transfer in the case of the bacteriophage MuA transposase (23; see also Harshey, this volume), perhaps indicating that this step where the nucleophile is a 3′-OH group is less stringent.…”

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

Calcium Inhibition of Ribonuclease H1 Two-Metal Ion Catalysis

Cited by 56 publications

References 93 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

Cation trafficking propels RNA hydrolysis

Mechanisms of DNA Transposition

Contact Info

Product

Resources

About