Terpenes constitute the largest and structurally most diverse natural product family. Most terpenoids exhibit a stereochemically complex macrocyclic core, which is generated by C–C bond forming of aliphatic oligo-prenyl precursors. This reaction is catalysed by terpene synthases (TPSs), which are capable of chaperoning highly reactive carbocation intermediates through an enzyme-specific reaction. Due to the instability of carbocation intermediates, the proteins’ structural dynamics and enzyme:substrate interactions during TPS catalysis remain elusive. Here, we present the structure of the diterpene synthase CotB2, in complex with an in crystallo cyclised abrupt reaction product and a substrate-derived diphosphate. We captured additional snapshots of the reaction to gain an overview of CotB2’s catalytic mechanism. To enhance insights into catalysis, structural information is augmented with multiscale molecular dynamic simulations. Our data represent fundamental TPS structure dynamics during catalysis, which ultimately enable rational engineering towards tailored terpene macrocycles that are inaccessible by conventional chemical synthesis.
A key question concerning the catalytic cycle of dihydrofolate reductase (DHFR) is whether the Met loop is dynamically coupled to the chemical step during catalysis. A more basic, yet unanswered question is whether the Met loop adopts a closed conformation during the chemical hydride transfer step. To examine the most likely conformation of the Met loop during the chemical step, we studied the hydride transfer in wild type (WT) DHFR using hybrid quantum mechanics-molecular mechanics free energy simulations with the Met loop in a closed and disordered conformation. Additionally, we investigated three mutant forms (I14; = Val, Ala, Gly) of the enzyme that have increased active site flexibility and donor-acceptor distance dynamics in closed and disordered Met loop states. We found that the conformation of the Met loop has a dramatic effect on the ordering of active site hydration, although the Met loop conformation only has a moderate effect on the hydride transfer rate and donor-acceptor distance dynamics. Finally, we evaluated the p of the substrate N5 position in closed and disordered Met loop states and found a strong correlation between N5 basicity and the conformation of the Met loop.
This paper deals with the effect of different size gold nanoparticles on the fluidity of lipid membrane at different regions of the bilayer. To investigate this, we have considered significantly large bilayer leaflets and incorporated only one nanoparticle each time, which was subjected to all atomistic molecular dynamics simulations. We have observed that, lipid molecules located near to the gold nanoparticle interact directly with it, which results in deformation of lipid structure and slower dynamics of lipid molecules. However, lipid molecules far away from the interaction site of the nanoparticle get perturbed, which gives rise to increase in local ordering of the lipid domains and decrease in fluidity. The bilayer thickness and area per head group in this region also get altered. Similar trend, but with different magnitude is also observed when different size nanoparticle interact with the bilayer.
Enzymes play a pivotal role in all biological systems. These biomachines are the most effective catalysts known, dramatically enhancing the rate of reactions by more than 10 orders of magnitude relative to the uncatalyzed reactions in solution. Predicting the correct, mechanistically appropriate binding modes for substrate and product, as well as all reaction intermediates and transition states, along a reaction pathway is immensely challenging and remains an unsolved problem. In the present work, we developed an effective methodology for identifying probable binding modes of multiple ligand states along a reaction coordinate in an enzyme active site. The program is called EnzyDock and is a CHARMM-based multistate consensus docking program that includes a series of protocols to predict the chemically relevant orientation of substrate, reaction intermediates, transition states, product, and inhibitors. EnzyDock is based on simulated annealing molecular dynamics and Monte Carlo sampling and allows ligand, as well as enzyme side-chain and backbone flexibility. The program can employ many user-defined constraints and restraints and classical force field potentials, as well as a range of hybrid quantum mechanics-molecular mechanics potentials. Herein, we apply EnzyDock to several different kinds of problems. First, we study two terpene synthase reactions, namely bornyl diphosphate synthase and the bacterial diterpene synthase CotB2. Second, we use EnzyDock to predict reaction coordinate states in a pair of Diels–Alder reactions in the enzymes spirotetronate AbyU and LepI. Third, we study a couple of racemases: the cofactor-dependent serine racemase and the cofactor independent proline racemase. Finally, we study several cases of covalent docking involving the Michael addition reaction. For all systems we predict binding modes that are consistent with available experimental observations, as well as with theoretical modeling studies from the literature. EnzyDock provides a platform for generating mechanistic insight into enzyme reactions, useful and reliable starting points for in-depth multiscale modeling projects, and rational design of noncovalent and covalent enzyme inhibitors.
In the present study, we address the effect of active site structure and dynamics of different dihydrofolate reductase (DHFR) isoforms on the pK a of the bound substrate 7,8-dihydrofolate, in an attempt to understand possible evolutionary trends. We apply a hybrid QM/MM free energy perturbation method to estimate the pK a of the N5 position of the bound substrate. We observe a gradual increase in N5 basicity as we move from primitive to more evolved DHFR isoforms. Structural analysis of these isoforms reveals a gradual sequestering of water molecules from the active site in the more evolved enzymes, thereby modulating the local dielectric environment near the substrate. Furthermore, the present study reveals a clear correlation between active site hydration and the N5 pK a of the substrate. We emphasize the role of the M20 loop in controlling the active site hydration level, via a preorganized active site with a more hydrophobic environment and reduced loop flexibility as evolution progresses from bacterial to the human enzyme.
The current study deals with the self-assembly of phospholipids on flat supports using the Martini coarse grain model. We reported here the effect of the hydrophilic and hydrophobic nature of the solid supports on the lipid self-assembly. The hydrophilic and hydrophobic supports were modeled on the basis of water droplet simulations. The present work addresses the self-assembly mechanism of lipids on eight different supports with different strengths of hydrophilicity and hydrophobicity. We demonstrated how interplay between the interactions of lipid and water with the support can guide the lipid self-assembly process. Thereafter, we calculated the energetics of the components of the system to quantify the competitions between water and a lipid head-group with hydrophilic supports. Finally, the properties of the self-assembled bilayers were also analyzed and reported here.
Glycerol-3-phosphate dehydrogenase is a biomedically important enzyme that plays a crucial role in lipid biosynthesis. It is activated by a ligand-gated conformational change that is necessary for the enzyme to reach a catalytically competent conformation capable of efficient transition-state stabilization. While the human form ( hl GPDH) has been the subject of extensive structural and biochemical studies, corresponding computational studies to support and extend experimental observations have been lacking. We perform here detailed empirical valence bond and Hamiltonian replica exchange molecular dynamics simulations of wild-type hl GPDH and its variants, as well as providing a crystal structure of the binary hl GPDH·NAD R269A variant where the enzyme is present in the open conformation. We estimated the activation free energies for the hydride transfer reaction in wild-type and substituted hl GPDH and investigated the effect of mutations on catalysis from a detailed structural study. In particular, the K120A and R269A variants increase both the volume and solvent exposure of the active site, with concomitant loss of catalytic activity. In addition, the R269 side chain interacts with both the Q295 side chain on the catalytic loop, and the substrate phosphodianion. Our structural data and simulations illustrate the critical role of this side chain in facilitating the closure of hl GPDH into a catalytically competent conformation, through modulating the flexibility of a key catalytic loop (292-LNGQKL-297). This, in turn, rationalizes a tremendous 41,000 fold decrease experimentally in the turnover number, k cat , upon truncating this residue, as loop closure is essential for both correct positioning of key catalytic residues in the active site, as well as sequestering the active site from the solvent. Taken together, our data highlight the importance of this ligand-gated conformational change in catalysis, a feature that can be exploited both for protein engineering and for the design of allosteric inhibitors targeting this biomedically important enzyme.
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