10 research outputs found

    Molecular Dynamics Simulations Elucidate Conformational Dynamics Responsible for the Cyclization Reaction in TEAS

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    The Mg-dependent 5-epi-aristolochene synthase from <i>Nicotiana tabacum</i> (called TEAS) could catalyze the linear farnesyl pyrophosphate (FPP) substrate to form bicyclic hydrocarbon 5-epi-aristolochene. The cyclization reaction mechanism of TEAS was proposed based on static crystal structures and quantum chemistry calculations in a few previous studies, but substrate FPP binding kinetics and protein conformational dynamics responsible for the enzymatic catalysis are still unclear. Herein, by elaborative and extensive molecular dynamics simulations, the loop conformation change and several crucial residues promoting the cyclization reaction in TEAS are elucidated. It is found that the unusual noncatalytic NH<sub>2</sub>-terminal domain is essential to stabilize Helix-K and the adjoining J-K loop of the catalytic COOH-terminal domain. It is also illuminated that the induce-fit J-K/A-C loop dynamics is triggered by Y527 and the optimum substrate binding mode in a ā€œU-shapeā€ conformation. The U-shaped ligand binding pose is maintained well with the cooperative interaction of the three Mg<sup>2+</sup>-containing coordination shell and conserved residue W273. Furthermore, the conserved Arg residue pair R264/R266 and aromatic residue pair Y527/W273, whose spatial orientations are also crucial to promote the closure of the active site to a hydrophobic pocket, as well as to form Ļ€-stacking interactions with the ligand, would facilitate the carbocation migration and electrophilic attack involving the catalytic reaction. Our investigation more convincingly proves the greater roles of the protein local conformational dynamics than do hints from the static crystal structure observations. Thus, these findings can act as a guide to new protein engineering strategies on diversifying the sesquiterpene products for drug discovery

    Biosynthesis of Spinosyn A: A [4 + 2] or [6 + 4] Cycloaddition?

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    SpnF, one of the Dielsā€“Alderases, produces spinosyn A, and previous work demonstrated that its sole function is to catalyze the [4 + 2] cycloaddition (Fage, C. D.; et al. Nat. Chem. Biol. 2015, 11, 256āˆ’258). Furthermore, the potential existence of a [6 + 4] cycloaddition bifurcation from previous theoretical calculations on the nonenzyme model (Patel, A.; et al. J. Am. Chem. Soc. 2016, 138, 3631āˆ’3634) shows that the exact mechanism of SpnF becomes even more interesting as well as now being controversial. In the present work, QMĀ­(DFT)/MM MD simulations on the full enzyme model revealed three significant residues that collaborate with other residues to control the direction of the cycloaddition, namely, Tyr23, Thr196, and Trp256. These residues force the substrate into a reactive conformation that causes the cycloaddition reaction to proceed through a [4 + 2] pathway instead of the [6 + 4] one. The mechanistic insights deciphered here are fundamentally important for the rational design of Dielsā€“Alderases and biomimetic syntheses

    A Comprehensive Understanding of Enzymatic Catalysis by Hydroxynitrile Lyases with <i>S</i> Stereoselectivity from the Ī±/Ī²-Hydrolase Superfamily: Revised Role of the Active-Site Lysine and Kinetic Behavior of Substrate Delivery and Sequential Product Release

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    The highly homologous hydroxynitrile lyases from Manihot esculent (<i>Me</i>HNL) and Hevea brasiliensis (<i>Hb</i>HNL) both belong to the Ī±/Ī²-hydrolase superfamily, and they convert cyanohydrins into the corresponding ketone (aldehyde) and hydrocyanic acid, which is important for biosynthesis for carbonā€“carbon formation. On the basis of extensive MM and ab initio QM/MM MD simulations, one-dimensional and two-dimensional free energy profiles on the whole enzymatic catalysis by <i>Me</i>HNL have been explored, and the effects of key residues around the channel on the delivery of substrate and product have been discussed. The residue Trp128 plays an important gate-switching role to manipulate the substrate access to the active site and product release. In particular, the release of acetone and HCN has been first detected to follow a stepwise mechanism. The release of HCN is quite facile, while the escape of acetone experiences a barrier of āˆ¼10 kcal/mol. The chemical reaction is an endergonic process with a free energy barrier of āˆ¼17.1 kcal/mol, which dominates the entire enzymatic efficiency. Such energy costs can be compensated by the remarkable energy release during the initial substrate binding. Here the carbonā€“carbon cleavage is the rate-determining step, which differs from that of <i>Hb</i>HNL. The protonation state of Lys237 plays an important role in carbonā€“carbon bond cleavage by restoring the Ser80Ala mutant system to the wild system, which explains the discrepancy between <i>Me</i>HNL and <i>Hb</i>HNL at the molecular or atomic scale. The present results provide a basis for understanding the similarity and difference in the enzymatic catalysis by <i>Me</i>HNL and <i>Hb</i>HNL

    Protonation-Dependent Diphosphate Cleavage in FPP Cyclases and Synthases

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    The cleavage of the magnesium-assisted diphosphate group (the PPi group) is one significant and prevalent rate-limiting step triggering the enzyme catalysis synthesis of terpenoid natural products. However, the PPi cleavage procedure has been rarely studied in most theoretical research of the terpenoid biosynthetic mechanism. In this work, QMĀ­(DFT)/MM MD simulations were employed to illuminate the detailed PPi cleavage mechanism in three different enzyme systems (ATAS, TEAS, and FPPS). We found that the most rational protonation state of the PPi group is highly dependent on the Mg<sup>2+</sup> coordination modes and the enzyme classes. The deprotonation of PPi is favorable for triggering the catalysis reaction in ATAS, while monoprotonation in FPPS and biprotonation in TEAS are advantageous. As a result, similar PPi cleavage occurs by means of nucleophilic substitution reactions in TEAS/FPPS/ATAS but presents an S<sub>N</sub>1, S<sub>N</sub>2, and borderline mechanism, respectively. Finally, the alternative functions of PPi protonation and Mg<sup>2+</sup> coordination modes are discussed

    QM/MM and MM MD Simulations on the Pyrimidine-Specific Nucleoside Hydrolase: A Comprehensive Understanding of Enzymatic Hydrolysis of Uridine

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    The pyrimidine-specific nucleoside hydrolase Yeik (CU-NH) from Escherichia coli cleaves the N-glycosidic bond of uridine and cytidine with a 10<sup>2</sup>ā€“10<sup>4</sup>-fold faster rate than that of purine nucleoside substrates, such as inosine. Such a remarkable substrate specificity and the plausible hydrolytic mechanisms of uridine have been explored by using QM/MM and MM MD simulations. The present calculations show that the relatively stronger hydrogen-bond interactions between uridine and the active-site residues Gln227 and Tyr231 in CU-NH play an important role in enhancing the substrate binding and thus promoting the N-glycosidic bond cleavage, in comparison with inosine. The estimated energy barrier of 30 kcal/mol for the hydrolysis of inosine is much higher than 22 kcal/mol for uridine. Extensive MM MD simulations on the transportation of substrates to the active site of CU-NH indicate that the uridine binding is exothermic by āˆ¼23 kcal/mol, more remarkable than inosine (āˆ¼12 kcal/mol). All of these arise from the noncovalent interactions between the substrate and the active site featured in CU-NH, which account for the substrate specificity. Quite differing from other nucleoside hydrolases, here the enzymatic N-glycosidic bond cleavage of uridine is less influenced by its protonation

    Concerted Cyclization of Lanosterol Cā€‘Ring and Dā€‘Ring Under Human Oxidosqualene Cyclase Catalysis: An ab Initio QM/MM MD Study

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    Human oxidosqualene cyclase (OSC) is one key enzyme in the biosynthesis of cholesterol. It can catalyze the linear-chain 2,3-oxidosqualene to form lanosterol, the tetracyclic (6ā€“6ā€“6ā€“5 members for Aā€“Bā€“Cā€“D rings) cholesterol precursor. It also has been treated as a novel antihyperlipidemia target. In addition, the structural diversity of cyclic terpenes in plants originates from the cyclization of 2,3-oxidosqualene. The enzyme catalytic mechanism is considered to be one of the most complicated ones in nature, and there are a lot of controversies about the mechanism in the past half a century. Herein, state-of-the-art ab initio QM/MM MD simulations are employed to investigate the detailed cyclization mechanism of C-ring and D-ring formation. Our study reveals that the C and D rings are formed near-synchronously from a stable ā€œ6ā€“6ā€“5ā€ ring intermediate. Interestingly, the transition state of this concerted reaction presents a ā€œ6ā€“6-6ā€ structure motif, while this unstable ā€œ6ā€“6-6ā€ structure in our simulations is thought to be a stable intermediate state in most previous hypothetical mechanisms. Furthermore, as the tailed side chain of 2,3-oxidosqualene shows a Ī² conformation while it is Ī± conformation in lanosterol, finally, it is observed that the rotatable ā€œtailā€ chain prefers to transfer Ī² conformation to Ī± conformation at the ā€œ6ā€“6ā€“5ā€ intermediate state

    Mechanistic Insights into the Rate-Limiting Step in Purine-Specific Nucleoside Hydrolase

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    A full enzymatic catalysis cycle in the inosineā€“adenosineā€“guanosine specific nucleoside hydrolase (IAG-NH) was assumed to be comprised of four steps: substrate binding, chemical reaction, base release, and ribose release. Nevertheless, the mechanistic details for the rate-limiting step of the entire enzymatic reaction are still unknown, even though the ribose release was likely to be the most difficult stage. Based on state-of-the-art quantum mechanics and molecular mechanics (QM/MM) molecular dynamics (MD) simulations, the ribose release process can be divided into two steps: ā€œribose dissociationā€ and ā€œribose releaseā€. The ā€œribose dissociationā€ includes ā€œcleavageā€ and ā€œexchangeā€ stages, in which a metastable 6-fold intermediate will recover to an 8-fold coordination shell of Ca<sup>2+</sup> as observed in <i>apo</i>- IAG-NH. Extensive random acceleration molecular dynamics and MD simulations have been employed to verify plausible release channels, and the estimated barrier for the rate-determining step of the entire reaction is 13.0 kcal/mol, which is comparable to the experimental value of 16.7 kcal/mol. Moreover, the gating mechanism arising from loop1 and loop2, as well as key residues around the active pocket, has been found to play an important role in manipulating the ribose release

    Computational Design of a Time-Dependent Histone Deacetylase 2 Selective Inhibitor

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    Development of isoform-selective histone deacetylase (HDAC) inhibitors is of great biological and medical interest. Among 11 zinc-dependent HDAC isoforms, it is particularly challenging to achieve isoform inhibition selectivity between HDAC1 and HDAC2 due to their very high structural similarities. In this work, by developing and applying a novel de novo reaction-mechanism-based inhibitor design strategy to exploit the reactivity difference, we have discovered the first HDAC2-selective inhibitor, Ī²-hydroxymethyl chalcone. Our bioassay experiments show that this new compound has a unique time-dependent selective inhibition on HDAC2, leading to about 20-fold isoform-selectivity against HDAC1. Furthermore, our ab initio QM/MM molecular dynamics simulations, a state-of-the-art approach to study reactions in biological systems, have elucidated how the Ī²-hydroxymethyl chalcone can achieve the distinct time-dependent inhibition toward HDAC2

    Molecular Dynamics-Based Virtual Screening: Accelerating the Drug Discovery Process by High-Performance Computing

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    High-performance computing (HPC) has become a state strategic technology in a number of countries. One hypothesis is that HPC can accelerate biopharmaceutical innovation. Our experimental data demonstrate that HPC can significantly accelerate biopharmaceutical innovation by employing molecular dynamics-based virtual screening (MDVS). Without using HPC, MDVS for a 10K compound library with tens of nanoseconds of MD simulations requires years of computer time. In contrast, a state of the art HPC can be 600 times faster than an eight-core PC server is in screening a typical drug target (which contains about 40K atoms). Also, careful design of the GPU/CPU architecture can reduce the HPC costs. However, the communication cost of parallel computing is a bottleneck that acts as the main limit of further virtual screening improvements for drug innovations

    Molecular Dynamics-Based Virtual Screening: Accelerating the Drug Discovery Process by High-Performance Computing

    No full text
    High-performance computing (HPC) has become a state strategic technology in a number of countries. One hypothesis is that HPC can accelerate biopharmaceutical innovation. Our experimental data demonstrate that HPC can significantly accelerate biopharmaceutical innovation by employing molecular dynamics-based virtual screening (MDVS). Without using HPC, MDVS for a 10K compound library with tens of nanoseconds of MD simulations requires years of computer time. In contrast, a state of the art HPC can be 600 times faster than an eight-core PC server is in screening a typical drug target (which contains about 40K atoms). Also, careful design of the GPU/CPU architecture can reduce the HPC costs. However, the communication cost of parallel computing is a bottleneck that acts as the main limit of further virtual screening improvements for drug innovations
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