Computational Insights into the High-Fidelily Catalysis of Aminoacyl-tRNA Synthetases

Obtaining insights into the catalytic function of enzymes is an important area of research due to their widespread applications in the biotechnology and pharmaceutical industries. Among these enzymes, the aminoacyl-tRNA synthetases (aaRSs) are known for their remarkable fidelity in catalyzing the aminoacylation reactions of tRNA in protein biosynthesis. Despite the exceptional execution of this critical function, mechanistic details of the reactions catalyzed by aminoacyl-tRNA synthetases remain elusive demonstrating the obvious need to explore their remarkable chemistry. During the PhD studies reported in this thesis the mechanism of aminoacylation, pre-transfer editing and post-transfer editing catalyzed by different aaRS have been established using multi-scale computational enzymology. In the first two chapters a detailed information about aaRS and the addressed questions was given in addition to an overview of the used computational methodology currently used to investigate the enzymatic mechanisms. The aminoacylation mechanism of threonine by Threonyl-tRNA synthetases, glutamine by Glutaminyl-tRNA synthetases and glutamate by Glutamyl-tRNA synthetases have been clearly unveiled in chapter 3 and 4. Also, valuable information regarding the role of cofactors and active site residues has been obtained. While investigating the post-transfer editing mechanisms, which proceed in a remote and distinct active site, two different scenarios were experimentally suggested for two types of threonyltRNA synthetase species to correct the misacylation of the structurally related serine. We explored these two mechanisms as in chapters 5 and 6. Moreover, the synthetic site in which the aminoacylation reaction is catalyzed, is also responsible for a second type of proofreading reaction called pre-transfer editing mechanism. In chapter 7, this latter mechanism has been elucidated for both Seryl-tRNA synthetases and Isoleucyl-tRNA synthetases against their non-cognate substrates cysteine and valine, respectively. In chapter 8, an assessment QM/MM study using a variety of DFT functionals to represent the chemically active layer in aminoacylation mechanism of the unnatural amino acid ß-Hydroxynorvaline as catalyzed by Threonyl-tRNA synthetase has been carried out. Overall, it was found that substrateassisted mechanisms are a common pathway for these enzymes. One important application of such information is to establish the criteria required for any candidate to inhibit the catalytic functions of aaRS, which was applied in chapter 9 to screen potential competitive inhibitors able to efficiently block the bacterial Threonyl-tRNA synthetases. The investigations reported herein should provide atomistic details into the fundamental catalytic mechanisms of the ubiquitous and ancient aaRS enzymes. Consequently, they will also help enable a much-needed deeper understanding of the underlying chemical principles of catalysis in general

Insights from molecular dynamics on substrate binding and effects of active site mutations in Delta1-pyrroline-5-carboxylate dehydrogenase

The NAD+-dependent enzyme, 1-pyrroline-5-carboxylate dehydrogenase (P5CDH), has an important role in proline and hydroxyproline catabolism for humans. Specifically, this aldehyde dehydrogenase is responsible for the oxidation of both L-glutamate- -semialdehyde (GSA) and 4-erythro-hydroxy-L-glutamate- -semialdehyde (4-OH-GSA) to their respective L-glutamate product forms. We have performed a detailed molecular dynamics (MD) study of both the reactant and product complex structures of P5CDH to gain insights into ligand binding (i.e., GSA, 4-OH-GSA, NAD+, GLU) in the active site. Moreover, our investigations were further extended to examine the structural impact of S352L, S352A, and E314A mutations on the deficiency in the P5CDH enzymatic activity. Our in silico mutation analysis indicated that the conserved Glu447 has significantly shifted in both the S352L and E314A mutants, causing NAD+ to be displaced from its predictive orientation in the binding site and hence forming a catalytically inactive enzyme. However in the case of S352A, the catalytic site including the oxyanion hole and Cys348 remain virtually unchanged, and the coenzyme maintains its binding position

Substrate-Assisted and Enzymatic Pretransfer Editing of Nonstandard Amino Acids by Methionyl-tRNA Synthetase

Aminoacyl-tRNA synthetases (aaRSs) are cen- tral to a number of physiological processes, including protein biosynthesis. In particular, they activate and then transfer their corresponding amino acid to the cognate tRNA. This is achieved with a generally remarkably high fidelity by editing against incorrect standard and nonstandard amino acids. Using docking, molecular dynamics (MD), and hybrid quantum mechanical/molecular mechanics methods, we have inves- tigated mechanisms by which methionyl-tRNA synthetase (MetRS) may edit against the highly toxic, noncognate, amino acids homocysteine (Hcy) and its oxygen analogue, homo- serine (Hse). Substrate-assisted editing of Hcy-AMP in which its own phosphate acts as the mechanistic base occurs with a rate-limiting barrier of 98.2 kJ mol−1. This step corresponds to nucleophilic attack of the Hcy side-chain sulfur at its own carbonyl carbon (CCarb). In contrast, a new possible editing mechanism is identified in which an active site aspartate (Asp259) acts as the base. The rate-limiting step is now rotation about the substrate’s aminoacyl Cβ−Cγ bond with a barrier of 27.5 kJ mol−1, while for Hse-AMP, the rate-limiting step is cleavage of the CCarb−OP bond with a barrier of 30.9 kJ mol−1. A similarly positioned aspartate or glutamate also occurs in the homologous enzymes LeuRS, IleRS, and ValRS, which also discriminate against Hcy. Docking and MD studies suggest that at least in the case of LeuRS and ValRS, a similar editing mechanism may be possible

Roles of the Active Site Zn(II) and Residues in Substrate Discrimination by Threonyl-tRNA Synthetase: An MD and QM/MM Investigation

Threonyl-tRNA synthetase (ThrRS) is a Zn(II) containing enzyme that catalyzes the activation of threonine and its subsequent transfer to the cognate tRNA. This process is accomplished with remarkable fidelity, with ThrRS being able to discriminate its cognate substrate from similar analogues such as serine and valine. Molecular dynamics (MD) simulations and hybrid quantum mechanics/molecular mechanics (QM/MM) methods have been used to elucidate the role of Zn(II) in the aminoacylation mechanism of ThrRS. More specifically, the role of Zn(II) and active site residues in ThrRS’s ability to discriminate between its cognate substrate L-threonine and the noncognate L-serine, L-valine, and D-threonine has been examined. The present results suggest that a role of the Zn(II) ion, with its Lewis acidity, is to facilitate deprotonation of the side chain hydroxyl groups of the aminoacyl moieties of cognate Thr-AMP and noncognate Ser-AMP substrates. In their deprotonated forms, these substrates are able to adopt a conformation preferable for aminoacyl transfer from aa-AMP onto the Ado-3′OH of the tRNAThr cosubstrate. Relative to the neutral substrates, when the substrates are first deprotonated with the assistance of the Zn(II) ion, the barrier for the rate-limiting step is decreased significantly by 42.0 and 39.2 kJ mol−1 for L-Thr-AMP and L-Ser-AMP, respectively. An active site arginyl also plays a key role in stabilizing the buildup of negative charge on the substrate’s bridging phosphate oxygen during the mechanism. For the enantiomeric substrate analogue D-Thr-AMP, product formation is highly disfavored, and as a result, the reverse reaction has a very low barrier of 16.0 kJ mol−1

Enzymatic Post-Transfer Editing Mechanism of E. coli Threonyl-tRNA Synthetase (ThrRS): A Molecular Dynamics (MD) and Quantum Mechanics/Molecular Mechanics (QM/MM) Investigation

Threonyl-tRNA synthetase (ThrRS) is a class II aminoacyl-tRNA synthetase (aaRS), which are a ubiquitous family of enzymes that have a vital role in protein biosynthesis. In particular, it catalyzes the activation and subsequent aminoacylation of its corresponding tRNAThr. Because of the close structural and electronic similarity between its cognate substrate threonine and the noncognate serine, the catalytic aminoacylation site of ThrRS is not able to fully discriminate between them. In this study we have explored multiple possible post-transfer editing mechanisms for ThrRS from Escherichia coli. The editing site is known to contain two conserved histidyls (His73 and His186) and a cysteinyl (Cys182), all of which could act as the required mechanistic base. We have performed detailed molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) studies in which the protonation states of each of these residues was varied. Furthermore, using the various substrate-bound active site models obtained, we have examined previously proposed and alternative possible mechanisms for deaminoacylation of Ser-tRNAThr by ThrRS in which His73 or Cys182 acts as the base: 11 mechanisms in total. The present results suggest that the most feasible mechanism is obtained when both His73 and His186 are neutral, while the thiol of Cys182 is deprotonated and acts as a base. The resulting mechanism is found to occur in two steps. First, deprotonation of an active site water by the thiolate of Cys182 with its concomitant nucleophilic attack at the substrate’s Ccarb center occurs with a calculated free energy barrier of 9.9 kcal/mol. The subsequent, and overall rate-limiting step, is a water-meditated proton transfer from Lys156 onto the Ado763′-oxygen resulting in simultaneous cleavage of the Ado763′O−Ccarb bond with a free energy barrier of 20.8 kcal/mol

A water-mediated and substrate-assisted aminoacylation mechanism in the discriminating aminoacyl-tRNA synthetase GlnRS and non-discriminating GluRS

Glutaminyl-tRNA synthetase (GlnRS) catalyzes the aminoacylation of glutamine to the corresponding tRNAGln. However, most bacteria and all archaea lack GlnRS and thus an indirect noncanonical aminoacylation is required. With the assistance of a non-discriminating version of Glutamyl-tRNA synthetases (ND-GluRS) the tRNAGln is misaminoacylated by glutamate. In this study, we have computationally investigated the aminoacylation mechanism in GlnRS and ND-GluRS employing Molecular Dynamics (MD) simulations, Quantum Mechanics (QM) cluster and Quantum Mechanics/Molecular Mechanics (QM/MM) calculations. Our investigations demonstrated the feasibility of a water-mediated, substrate-assisted catalysis pathway with rate limiting steps occurring at energy barriers of 25.0 and 25.4 kcal mol−1 for GlnRS and ND-GluRS, respectively. A conserved lysine residue participates in a second proton transfer to facilitate the departure of the adenosine monophosphate (AMP) group. Thermodynamically stable (−29.9 and −9.3 kcal mol−1 for GlnRS and ND-GluRS) product complexes are obtained only when the AMP group is neutral

Insights from molecular dynamics on substrate binding and effects of active site mutations in Δ1-pyrroline-5-carboxylate dehydrogenase

The NAD+-dependent enzyme, 1 -pyrroline-5-carboxylate dehydrogenase (P5CDH), has an important role in proline and hydroxyproline catabolism for humans. Specifically, this aldehyde dehydrogenase is responsible for the oxidation of both L-glutamate--semialdehyde (GSA) and 4-erythro-hydroxy-L-glutamate--semialdehyde (4-OH-GSA) to their respective L-glutamate product forms. We have performed a detailed molecular dynamics (MD) study of both the reactant and product complex structures of P5CDH to gain insights into ligand binding (i.e., GSA, 4-OH-GSA, NAD+, GLU) in the active site. Moreover, our investigations were further extended to examine the structural impact of S352L, S352A, and E314A mutations on the deficiency in the P5CDH enzymatic activity. Our in silico mutation analysis indicated that the conserved Glu447 has significantly shifted in both the S352L and E314A mutants, causing NAD+ to be displaced from its predictive orientation in the binding site and hence forming a catalytically inactive enzyme. However in the case of S352A, the catalytic site including the oxyanion hole and Cys348 remain virtually unchanged, and the coenzyme maintains its binding position. --- La 1 -pyrroline-5-carboxylate déshydrogénase (P5CDH), une enzyme dépendante du NAD+, joue un rôle important dans le catabolisme de la proline et de l’hydroxyproline chez l’humain. En particulier, cette aldéhyde déshydrogénase est responsable de l’oxydation du L-glutamate--semialdéhyde (GSA) et du 4-érythro-hydroxy-L-glutamate--semialdéhyde (4-OH-GSA) en leurs formes respectives du L-glutamate. Nous avons réalisé une étude détaillée de dynamique moléculaire (DM) portant sur les structures des réactifs (c.-a`-d. le GSA, le 4-OH-GSA, le NAD+ et le GLU) et celles de leurs complexes avec la P5CDH en vue de mieux comprendre la liaison du ligand au site actif. De plus, nous avons approfondi nos recherches afin d’examiner l’incidence structurale des mutations S352L, S352A et E314A sur la réduction de l’activité enzymatique de la P5CDH. Notre analyse in silico des mutations a montré que le résidu Glu447 conservé dans les mutants S352L et E314A s’est considérablement déplacé, ce qui a entraîné un changement de position du NAD+ par rapport a` son orientation prévue dans le site de liaison, et par conséquent, la formation d’une enzyme inactive sur le plan catalytique. Toutefois, dans le cas de la mutation S352A, le site catalytique comprenant le trou oxyanion et le résidu Cys348 demeure essentiellement inchangé, et la coenzyme conserve la position de sa liaison. [Traduit par la Rédaction

An assessment to evaluate the validity of different methods for the description of some corrosion inhibitors

New research and development efforts using computational chemistry in studying an assessment of the validity of different quantum chemical methods to describe the molecular and electronic structures of some corrosion inhibitors were introduced. The standard and the highly accurate CCSD method with 6-311++G(d,p), ab initio calculations using the HF/6-31G++(d,p) and MP2 with 6-311G(d,p), 6-31++G(d,p), and 6-311++G(2df,p) methods as well as DFT method at the B3LYP, BP86, B3LYP*, M06L, and M062x/6-31G++(d,p) basis set level were performed on some triazole derivatives and sulfur containing compounds used as corrosion inhibitors. Quantum chemical parameters, such as the energy of the highest occupied molecular orbital energy (E(HOMO)), the energy of the lowest unoccupied molecular orbital energy (E(LUMO)), energy gap (ΔE), dipole moment (μ), sum of total negative charges (TNC), chemical potential (Pi), electronegativity (χ), hardness (η), softness (σ), local softness (s), Fukui functions (f (+),f (-)), electrophilicity (ω), the total energy change (∆E(T)) and the solvation energy (S.E), were calculated. Furthermore, the accuracy and the applicability of these methods were estimated relative to the highest accuracy and standard CCSD with 6-311++G(d,p) method. Good correlations between the quantum chemical parameters and the corresponding inhibition efficiency (IE%) were found

Roles of the Active Site Zn(II) and Residues in Substrate Discrimination by Threonyl-tRNA Synthetase: An MD and QM/MM Investigation

Threonyl-tRNA synthetase (ThrRS) is a Zn­(II) containing enzyme that catalyzes the activation of threonine and its subsequent transfer to the cognate tRNA. This process is accomplished with remarkable fidelity, with ThrRS being able to discriminate its cognate substrate from similar analogues such as serine and valine. Molecular dynamics (MD) simulations and hybrid quantum mechanics/molecular mechanics (QM/MM) methods have been used to elucidate the role of Zn­(II) in the aminoacylation mechanism of ThrRS. More specifically, the role of Zn­(II) and active site residues in ThrRS’s ability to discriminate between its cognate substrate l-threonine and the noncognate l-serine, l-valine, and d-threonine has been examined. The present results suggest that a role of the Zn­(II) ion, with its Lewis acidity, is to facilitate deprotonation of the side chain hydroxyl groups of the aminoacyl moieties of cognate Thr-AMP and noncognate Ser-AMP substrates. In their deprotonated forms, these substrates are able to adopt a conformation preferable for aminoacyl transfer from aa-AMP onto the Ado-3′OH of the tRNA^Thr cosubstrate. Relative to the neutral substrates, when the substrates are first deprotonated with the assistance of the Zn­(II) ion, the barrier for the rate-limiting step is decreased significantly by 42.0 and 39.2 kJ mol^–1 for l-Thr-AMP and l-Ser-AMP, respectively. An active site arginyl also plays a key role in stabilizing the buildup of negative charge on the substrate’s bridging phosphate oxygen during the mechanism. For the enantiomeric substrate analogue d-Thr-AMP, product formation is highly disfavored, and as a result, the reverse reaction has a very low barrier of 16.0 kJ mol^–1

Unraveling the Critical Role Played by Ado762′OH in the Post-Transfer Editing by Archaeal Threonyl-tRNA Synthetase

Archaeal threonyl-tRNA synthetase (ThrRS) possesses an editing active site wherein tRNAThr that has been misaminoacylated with serine (i.e., Ser-tRNAThr) is hydrolytically cleaved to serine and tRNAThr. It has been suggested that the free ribose sugar hydroxyl of Ado76 of the tRNAThr(Ado762′OH) is the mechanistic base, promoting hydrolysis by orienting a nucleophilic water near the scissile Ser-tRNAThr ester bond. We have performed a computational study, involving molecular dynamics (MD) and hybrid ONIOM quantum mechanics/molecular mechanics (QM/MM) methods, considering all possible editing mechanisms to gain an understanding of the role played by Ado762′OH group. More specifically, a range of concerted or stepwise mechanisms involving four-, six-, or eight-membered transition structures (total of seven mechanisms) were considered. In addition, these seven mechanisms were fully optimized using three different DFT functionals, namely, B3LYP, M06-2X, and M06-HF. The M06-HF functional gave the most feasible energy barriers followed by the M06-2X functional. The most favorable mechanism proceeds stepwise through two six-membered ring transition states in which the Ado762′OH group participates, overall, as a shuttle for the proton transfer from the nucleophilic H2O to the bridging oxygen (Ado763′O) of the substrate. More specifically, in the first step, which has a barrier of 25.9 kcal/mol, the Ado762′-OH group accepts a proton from the attacking nucleophilic water while concomitantly transferring its proton onto the substrates C–Ocarb center. Then, in the second step, which also proceeds with a barrier of 25.9 kcal/mol, the Ado762′-OH group transfers its proton on the adjacent Ado763′-oxygen, cleaving the scissile Ccarb–O3′Ado76 bond, while concomitantly accepting a proton from the previously formed C–OcarbH group