A physico-chemical rationale for the varied catalytic efficiency in RNase J paralogues DOI Creative Commons

A. K. Singh,

Chinnasamy Kalaiarasi, Nikhil Pahelkar

et al.

Journal of Biological Chemistry, Journal Year: 2024, Volume and Issue: unknown, P. 108152 - 108152

Published: Dec. 1, 2024

Paralogues of the bifunctional nuclease, Ribonuclease J (RNase J) demonstrate varied catalytic efficiencies despite extensive sequence and structural similarity. Of two S. aureus RNase paralogues, J1 is substantially more active than J2. Mutational analysis site residues revealed that only H80 E166 were critical for nuclease activity. Electronic properties further evaluated using density functional theory in conjunction with molecular mechanics. This suggested multiple at can function as Lewis base or acid The bond dissociation energy, on other hand, Mn ion J2, located a structurally identical location to J1, crucial overall integrity. Structures mutant enzymes lacking metal seen adopt different orientation between substrate binding domain wild-type A surprising finding was J2 H78A five-fold wildtype enzyme. Structural biochemical experiments performed light this observation mechanism distinct from both two-metal one-metal reaction mechanisms proposed nucleases. Different activity levels paralogues thus be ascribed diversity mechanisms.

Language: Английский

Catching CRISPR-Cas9 in Action DOI
Yingjie Chen, Yuanhao Li, Penghai Li

et al.

Journal of Chemical Theory and Computation, Journal Year: 2025, Volume and Issue: unknown

Published: May 5, 2025

CRISPR-Cas9 has revolutionized genome editing, yet its structural dynamics and functional properties remain incompletely understood, partly due to limited atomic-level characterization of active conformation with a full R-loop. Capitalizing on recent advances in Cas9 determination, we constructed catalytic-state model bound bona fide R-loop performed an integrated computational investigation. Our molecular simulations reveal substantial conformational heterogeneity the PAM (protospacer-adjacent motif)-distal nontarget DNA strand adjacent regions, leading dynamically fluctuating interactions, thereby challenging experimental resolution complex. Comparative analysis highlights barrier restricting final activation HNH nuclease domain, suggesting that strategic modulation interactions two sides could enhance cleavage efficiency. Furthermore, quantum mechanics/molecular mechanics indicate H983 protonated at Nε, RuvC domain favors phosphate-mediated over histidine-mediated pathway for cleavage. Additionally, identify alternative HNH-mediated target pathway, involving water nucleophile aligned 5' side scissile phosphate. Inspired by basic residue ladder observed RuvC, propose extending similar strengthen binding catalytic activity. study provides critical insights into structure, dynamics, catalysis, laying foundation rational design next-generation systems optimized specificity-efficiency balance.

Language: Английский

Citations

0
Mechanism of Nucleic Acid Phosphodiester Bond Cleavage by Human Endonuclease V: MD and QM/MM Calculations Reveal a Versatile Metal Dependence DOI
Rajwinder Kaur, Dylan J. Nikkel, Stacey D. Wetmore

et al.

The Journal of Physical Chemistry B, Journal Year: 2024, Volume and Issue: 128(39), P. 9455 - 9469

Published: Sept. 23, 2024

Human endonuclease V (EndoV) catalytically removes deaminated nucleobases by cleaving the phosphodiester bond as part of RNA metabolism. Despite being implicated in several diseases (cancers, cardiovascular diseases, and neurological disorders) potentially a useful tool biotechnology, details human EndoV catalytic pathway remain unclear due to limited experimental information beyond crystal structure apoenzyme select mutational data. Since mechanistic understanding is critical for further deciphering central roles expanding applications medicine molecular dynamics (MD) simulations quantum mechanics/molecular mechanics (QM/MM) calculations were used unveil atomistic pathway. Due controversies surrounding number metals required nuclease activity, enzyme-substrate models with different numbers active site various metal-substrate binding configurations built based on structural data other nucleases. Subsequent MD revealed stability EndoV-substrate complex range metal architectures. Four unique pathways then characterized using QM/MM that vary (one versus two) modes substrate coordination [direct indirect (water-mediated)], mechanisms fully consistent structural, kinetic, related nucleases, including members family. Beyond uncovering key amino acids (D240 K155), our highlight while one essential enzyme can benefit from two presence suitable sites. By directly comparing one- two-metal-mediated P-O cleavage reactions within confines same site, work brings fresh perspective "number metals" controversy.

Language: Английский

Citations

2
The phosphodiester dissociative hydrolysis of a DNA model promoted by metal dications DOI
Valdemir dos Santos, Felipe Ribeiro,

C. Kim

et al.

Journal of Molecular Modeling, Journal Year: 2024, Volume and Issue: 30(11)

Published: Oct. 23, 2024

Language: Английский

Citations

0
How Can One Metal Power Nucleic Acid Phosphodiester Bond Cleavage by a Nuclease? Multiscale Computational Studies Highlight a Diverse Mechanistic Landscape DOI
Dylan J. Nikkel, Rajwinder Kaur, Stacey D. Wetmore

et al.

The Journal of Physical Chemistry B, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 25, 2024

Despite the remarkable resistance of nucleic acid phosphodiester backbone to degradation affording genetic stability, P–O bond must be broken during DNA repair and RNA metabolism, among many other critical cellular processes. Nucleases are powerful enzymes that can enhance uncatalyzed rate cleavage by up ∼1017-fold. most well accepted hydrolysis mechanism involving two metals (MA2+ activate a water nucleophile MB2+ stabilize leaving group), experimental evidence suggests some nucleases use single metal facilitate chemical step, controversial concept in literature. The present perspective uses case studies four (I-PpoI, APE1, bacterial human EndoV) highlight how computational approaches ranging from quantum mechanical (QM) cluster models molecular dynamics (MD) simulations combined mechanics-molecular mechanics (QM/MM) calculations reveal atomic level details necessary understand nuclease this difficult chemistry. representative showcase different amino residues (e.g., histidine, aspartate) fulfill role first (MA2+) two-metal-mediated mechanisms. Nevertheless, differences active site architectures afford diversity single-metal-mediated terms metal–substrate coordination, metal, identities general base. greater understanding catalytic mechanisms obtained body work reviewed used further explore progression diseases associated with (mis)activity development novel applications such as disease diagnostics, gene engineering, therapeutics.

Language: Английский

Citations

0
A physico-chemical rationale for the varied catalytic efficiency in RNase J paralogues DOI Creative Commons

A. K. Singh,

Chinnasamy Kalaiarasi, Nikhil Pahelkar

et al.

Journal of Biological Chemistry, Journal Year: 2024, Volume and Issue: unknown, P. 108152 - 108152

Published: Dec. 1, 2024

Paralogues of the bifunctional nuclease, Ribonuclease J (RNase J) demonstrate varied catalytic efficiencies despite extensive sequence and structural similarity. Of two S. aureus RNase paralogues, J1 is substantially more active than J2. Mutational analysis site residues revealed that only H80 E166 were critical for nuclease activity. Electronic properties further evaluated using density functional theory in conjunction with molecular mechanics. This suggested multiple at can function as Lewis base or acid The bond dissociation energy, on other hand, Mn ion J2, located a structurally identical location to J1, crucial overall integrity. Structures mutant enzymes lacking metal seen adopt different orientation between substrate binding domain wild-type A surprising finding was J2 H78A five-fold wildtype enzyme. Structural biochemical experiments performed light this observation mechanism distinct from both two-metal one-metal reaction mechanisms proposed nucleases. Different activity levels paralogues thus be ascribed diversity mechanisms.

Language: Английский

Citations

0