<i>Deinococcus radiodurans </i><scp>DR</scp>2231 is a two‐metal‐ion mechanism hydrolase with exclusive activity on d<scp>UTP</scp>

Mota, Cristiano; Gonçalves, Aldina; Sanctis, Daniele de

doi:10.1111/febs.13923

Cited by 4 publications

(23 citation statements)

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“… 137 MD simulations of DNA Pol η indicated a possible role in active site stabilization on the basis of the observed partial unfolding of the DNA substrate and consequent distortion of the Michaelis–Menten complex upon mutation of these residues. Mutagenesis 195 and computational 196 studies on another nucleotide‐processing metalloenzyme, all‐α dimeric deoxyuridine triphosphate nucleotidohydrolase, suggested that such residues contribute to specificity by stabilizing the correct substrate.…”

Section: Computational Studies Of Polymerase Catalytic Mechanism and ...mentioning

confidence: 99%

Computational investigations of polymerase enzymes: Structure, function, inhibition, and biotechnology

Geronimo

Vidossich

Donati

et al. 2021

WIREs Comput Mol Sci

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DNA and RNA polymerases (Pols) are central to life, health, and biotechnology because they allow the flow of genetic information in biological systems. Importantly, Pol function and (de)regulation are linked to human diseases, notably cancer (DNA Pols) and viral infections (RNA Pols) such as COVID‐19. In addition, Pols are used in various applications such as synthesis of artificial genetic polymers and DNA amplification in molecular biology, medicine, and forensic analysis. Because of all of this, the field of Pols is an intense research area, in which computational studies contribute to elucidating experimentally inaccessible atomistic details of Pol function. In detail, Pols catalyze the replication, transcription, and repair of nucleic acids through the addition, via a nucleotidyl transfer reaction, of a nucleotide to the 3′‐end of the growing nucleic acid strand. Here, we analyze how computational methods, including force‐field‐based molecular dynamics, quantum mechanics/molecular mechanics, and free energy simulations, have advanced our understanding of Pols. We examine the complex interaction of chemical and physical events during Pol catalysis, like metal‐aided enzymatic reactions for nucleotide addition and large conformational rearrangements for substrate selection and binding. We also discuss the role of computational approaches in understanding the origin of Pol fidelity—the ability of Pols to incorporate the correct nucleotide that forms a Watson–Crick base pair with the base of the template nucleic acid strand. Finally, we explore how computations can accelerate the discovery of Pol‐targeting drugs and engineering of artificial Pols for synthetic and biotechnological applications. This article is categorized under: Structure and Mechanism > Reaction Mechanisms and Catalysis Structure and Mechanism > Computational Biochemistry and Biophysics Software > Molecular Modeling

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Section: Computational Studies Of Polymerase Catalytic Mechanism and ...mentioning

confidence: 99%

Computational investigations of polymerase enzymes: Structure, function, inhibition, and biotechnology

Geronimo

Vidossich

Donati

et al. 2021

WIREs Comput Mol Sci

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“…Intriguingly, recent experimental data have shown that the enzyme DR2231, a dimeric all-α dUTPase from Deinococcus radiodurans, selectively hydrolyzes only the dUTP substrate, in contrast to most members of the superfamily, which can unselectively hydrolyze both dUDP and dUTP . However, given that experimental evidence suggests binding of dUDP to the active site, DR2231’s specificity for dUTP is mechanistically unexplained. The availability of structural and kinetics data on DR2231 catalysis , provides an optimal opportunity to dissect the fundamental aspects of the enzymatic processing of nucleotides by dUTPases.…”

Section: Introductionmentioning

confidence: 99%

“…However, given that experimental evidence suggests binding of dUDP to the active site, DR2231’s specificity for dUTP is mechanistically unexplained. The availability of structural and kinetics data on DR2231 catalysis , provides an optimal opportunity to dissect the fundamental aspects of the enzymatic processing of nucleotides by dUTPases.…”

Section: Introductionmentioning

confidence: 99%

“…The bimetallic catalytic center is surrounded by a number of second-shell positively charged residues, which are commonly found in the vicinity of the reactive site in nucleic-acid-processing enzymes . Importantly, mutagenesis studies showed that such second-shell basic residues (namely, Lys112, Lys122, and Lys125) are required for efficient catalysis in DR2231 . These residues are involved in the interactions with the dUTP phosphate tail, although their exact functional contribution to catalysis is unclear.…”

Section: Introductionmentioning

confidence: 99%

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Functional Implications of Second-Shell Basic Residues for dUTPase DR2231 Enzymatic Specificity

et al. 2020

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Nucleotide-processing enzymes are key players in biological processes. They often operate through high substrate specificity for catalysis. How such specificity is achieved is unclear. Here, we dealt with this question by investigating all-α dimeric deoxyuridine triphosphate nucleotidohydrolases (dUTPases). Typically, these dUTPases hydrolyze either dUTP or deoxyuridine diphosphate (dUDP) substrates. However, the dUTPase enzyme DR2231 from Deinococcus radiodurans selectively hydrolyzes dUTP only, and not dUDP. By means of extended classical molecular dynamics simulations and quantum chemical calculations, we show that DR2231 achieves this specificity for dUTP via second-shell basic residues that, together with the two catalytic magnesium ions, contribute to properly orienting the γ-phosphate of dUTP in a prereactive state. This allows a nucleophilic water to be correctly placed and activated in order to perform substrate hydrolysis. We show that this enzymatic mechanism is not viable when dUDP is bound to DR2231. Importantly, in several other dUTPases capable of hydrolyzing either dUTP or dUDP, we detected that active site second-shell basic residues are more in number, anchoring the β-phosphate of the nucleotide substrate too, in contrast to what is observed in DR2231. Thus, strategically located basic second-shell residues mediate precise reactant positioning at the catalytic site, determining substrate specificity in dUTPases and possibly in other structurally similar nucleotide-processing metalloenzymes.

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“…Mn ions are preferred for the enzymatic activities of RecJ [15] , MazG [83] , and PolX [79] , while Zn ions are involved in protein folding and substrate processing/binding by RNase J [72] , PprI [21] , RecOR [77] , DinB [78] , and LuxS [80] . Compared with E. coli homologues that mainly use Mg ions as cofactors, RecJ [15] , MazG [83] , MutS2 [84] and PolX [79] in D. radiodurans exhibited a strong preference for Mn over Mg, which might be due to the intrinsic properties of these two metal ions, e.g., the coordination geometry required for catalysis. Moreover, Mn was essential for the protein dimerization of RNase J from D. radiodurans [72] , which is likely to be involved in the balance of its exo- and endonucleolytic activity.…”

Section: Metalloenzymesmentioning

confidence: 99%

Structural features and functional implications of proteins enabling the robustness of Deinococcus radiodurans

Chen

Tang

Hua

et al. 2020

Computational and Structural Biotechnology Journal

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Deinococcus radiodurans can survive under extreme conditions, including high doses of DNA damaging agents and ionizing radiation, desiccation, and oxidative stress. Both the efficient cellular DNA repair machinery and antioxidation systems contribute to the extreme resistance of this bacterium, making it an ideal organism for studying the cellular mechanisms of environmental adaptation. The number of stress-related proteins identified in this bacterium has mushroomed in the past two decades. The newly identified proteins reveal both commonalities and diversity of structure, mechanism, and function, which impact a wide range of cellular functions. Here, we review the unique and general structural features of these proteins and discuss how these studies improve our understanding of the environmental stress adaptation mechanisms of D. radiodurans .

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Deinococcus radiodurans DR2231 is a two‐metal‐ion mechanism hydrolase with exclusive activity on dUTP

Abstract: Structural data are available in the PDB under the accession numbers 5HVA, 5HWU, 5HX1, 5HYL, 5I0J, 5HZZ, 5I0M.

Cited by 4 publications

References 47 publications

Computational investigations of polymerase enzymes: Structure, function, inhibition, and biotechnology

Computational investigations of polymerase enzymes: Structure, function, inhibition, and biotechnology

Functional Implications of Second-Shell Basic Residues for dUTPase DR2231 Enzymatic Specificity

Structural features and functional implications of proteins enabling the robustness of Deinococcus radiodurans

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