Second-Shell Basic Residues Expand the Two-Metal-Ion Architecture of DNA and RNA Processing Enzymes

Genna, Vito; Colombo, Matteo; Vivo, Marco De; Marcia, Marco

doi:10.1016/j.str.2017.11.008

Cited by 36 publications

(87 citation statements)

References 70 publications

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“…Thus, in silico modelling strongly suggests that polymerases will selectively incorporate only the S p P-alkyl-phNTP diastereoisomers, which preserves the critical interaction of the negatively charged pro R p -oxygen with the catalytically essential proximal Mg 2+ ion (Mg A ) in the polymerase active site28, 30. Indeed, this is consistent with previous results that suggested stereospecific incorporation of only the S p P-Met-phTTP by Terminal transferase21.…”

Section: Resultssupporting

confidence: 88%

“…Our theoretical investigations unequivocally showed that only the S p phATP substrates were able to productively engage (and remain) within the polymerase holoenzyme active site (thus forming the polymerase-primer-template complex). This is demonstrated by three different and well-established structural/chemical determinants we monitored along the 6 μ s of MD trajectory to evaluate the reactivity of the precatalytic complex; namely: i) the root-mean-square deviation (RMSD) of the incoming substrate measuring its stability within polymerase active site27, ii) the length between the -3’O - (-3’OH) nucleophile of the primer 3’-end and the α-Phosphorus of the incoming phATP assessing the formation of a prone-to-react Michaelis-Menten complex28 and iii) the ability to form Watson-Crick (WC) H-bonds to the instructive template base, a condition known to further assist the incorporation of the incoming nucleotide to the growing primer strand29, 30.…”

Section: Resultsmentioning

confidence: 99%

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A synthetic genetic polymer with an uncharged backbone chemistry based on alkyl phosphonate nucleic acids

et al. 2019

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The physicochemical properties of nucleic acids are dominated by their highly charged phosphodiester backbone chemistry. The polyelectrolyte structure decouples information content (base sequence) from bulk properties such as solubility and has been proposed as a defining trait of all informational polymers. However, this conjecture has not been tested experimentally. Here, we describe the encoded synthesis of a genetic polymer with an uncharged backbone chemistry: alkyl-phosphonate nucleic acids (phNA), in which the canonical, negatively charged phosphodiester is replaced by an uncharged P-alkyl-phosphonodiester backbone. Using synthetic chemistry and polymerase engineering, we describe the enzymatic, DNA-templated synthesis of P-methyl- and P-ethyl-phNAs, and the directed evolution of specific streptavidin-binding phNA aptamer ligands directly from random-sequence, mixed P-methyl- / P-ethyl-phNA repertoires. Our results establish a first example of the DNA-templated enzymatic synthesis and evolution of an uncharged genetic polymer and provide a foundational methodology for their exploration as a source of novel, functional molecules.

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Section: Resultssupporting

confidence: 88%

Section: Resultsmentioning

confidence: 99%

A synthetic genetic polymer with an uncharged backbone chemistry based on alkyl phosphonate nucleic acids

et al. 2019

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“…This residue is in close proximity to the branch site 39 , it interacts with the 5′-splice site, and it undergoes a rearrangement between the splicing steps 40 in a process that is modulated by protein subunits (i.e. Prp8, Prp16) 41,42 and potassium ions 43,44 . Such reorganization of G52 facilitates the release of the 5′-end of spliceosomal introns from the active site after the first splicing step, while also favoring the recruitment of the 3′-splice junction into the active site for the second step of splicing 40 .…”

Section: And Supplementarymentioning

confidence: 99%

Visualizing group II intron dynamics between the first and second steps of splicing

et al. 2020

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Group II introns are ubiquitous self-splicing ribozymes and retrotransposable elements evolutionarily and chemically related to the eukaryotic spliceosome, with potential applications as gene-editing tools. Recent biochemical and structural data have captured the intron in multiple conformations at different stages of catalysis. Here, we employ enzymatic assays, X-ray crystallography, and molecular simulations to resolve the spatiotemporal location and function of conformational changes occurring between the first and the second step of splicing. We show that the first residue of the highly-conserved catalytic triad is protonated upon 5'-splice-site scission, promoting a reversible structural rearrangement of the active site (toggling). Protonation and active site dynamics induced by the first step of splicing facilitate the progression to the second step. Our insights into the mechanism of group II intron splicing parallels functional data on the spliceosome, thus reinforcing the notion that these evolutionarily-related molecular machines share the same enzymatic strategy.

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“…Another intriguing aspect is the recurring presence of second-shell positively charged residues that surround the metal-aided catalytic site in nucleic-acid-processing enzyme. 3 During our extended MD simulations, we observed the interaction of Arg93, along the helical arch, with the terminal phosphate of the substrate. We structurally aligned hExo1 (PDB ID 5V0E) 23 with hFEN1 (PDB ID 5KSE), 34 and we noted that the terminal guanidine, amide, and amine groups of Arg93, Asn124 (in hExo1), and Lys132 (in hFEN1) residues were all within a sphere of ∼3 Å and close to the 5′ phosphate in the leaving group ( Figure S12 ).…”

Section: Discussionmentioning

confidence: 95%

Recruiting Mechanism and Functional Role of a Third Metal Ion in the Enzymatic Activity of 5′ Structure-Specific Nucleases

2020

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Enzymes of the 5′ structure-specific nuclease family are crucial for DNA repair, replication, and recombination. One such enzyme is the human exonuclease 1 (hExo1) metalloenzyme, which cleaves DNA strands, acting primarily as a processive 5′-3′ exonuclease and secondarily as a 5′-flap endonuclease. Recently, in crystallo reaction intermediates have elucidated how hExo1 exerts hydrolysis of DNA phosphodiester bonds. These hExo1 structures show a third metal ion intermittently bound close to the two-metal-ion active site, to which recessed ends or 5′-flap substrates bind. Evidence of this third ion has been observed in several nucleic-acid-processing metalloenzymes. However, there is still debate over what triggers the (un)binding of this transient third ion during catalysis and whether this ion has a catalytic function. Using extended molecular dynamics and enhanced sampling free-energy simulations, we observed that the carboxyl side chain of Glu89 (located along the arch motif in hExo1) flips frequently from the reactant state to the product state. The conformational flipping of Glu89 allows one metal ion to be recruited from the bulk and promptly positioned near the catalytic center. This is in line with the structural evidence. Additionally, our simulations show that the third metal ion assists the departure, through the mobile arch, of the nucleotide monophosphate product from the catalytic site. Structural comparisons of nuclease enzymes suggest that this Glu(Asp)-mediated mechanism for third ion recruitment and nucleic acid hydrolysis may be shared by other 5′ structure-specific nucleases.

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Second-Shell Basic Residues Expand the Two-Metal-Ion Architecture of DNA and RNA Processing Enzymes

Cited by 36 publications

References 70 publications

A synthetic genetic polymer with an uncharged backbone chemistry based on alkyl phosphonate nucleic acids

A synthetic genetic polymer with an uncharged backbone chemistry based on alkyl phosphonate nucleic acids

Visualizing group II intron dynamics between the first and second steps of splicing

Recruiting Mechanism and Functional Role of a Third Metal Ion in the Enzymatic Activity of 5′ Structure-Specific Nucleases

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