Investigation of Structural Dynamics of Enzymes and Protonation States of Substrates Using Computational Tools

Chang, Chia‐en A.; Huang, Yu‐ming M.; Mueller, Leonard J.; You, Wanli

doi:10.3390/catal6060082

Cited by 13 publications

(7 citation statements)

References 111 publications

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“…Elucidating the protonation states of the numerous titratable moieties in the TrpB active site at the various stages of catalysis is an active area of research . Reaction with nitrogen nucleophiles, such as indoline and 2-aminophenol, stalls the catalytic cycle of TrpS from Salmonella typhimurium ( St TrpS), enabling the structural analysis of quinonoid intermediates. − These studies have indicated that proton transfer between the phenolic group of PLP and the Schiff base may occur during the catalytic cycle.…”

Section: Introductionmentioning

confidence: 99%

Directed Evolution Mimics Allosteric Activation by Stepwise Tuning of the Conformational Ensemble

et al. 2018

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Allosteric enzymes contain a wealth of catalytic diversity that remains distinctly underutilized for biocatalysis. Tryptophan synthase is a model allosteric system and a valuable enzyme for the synthesis of noncanonical amino acids (ncAA). Previously, we evolved the β-subunit from Pyrococcus furiosus, PfTrpB, for ncAA synthase activity in the absence of its native partner protein PfTrpA. However, the precise mechanism by which mutation activated TrpB to afford a stand-alone catalyst remained enigmatic. Here, we show that directed evolution caused a gradual change in the rate-limiting step of the catalytic cycle. Concomitantly, the steady-state distribution of the intermediates shifts to favor covalently bound Trp adducts, which have increased thermodynamic stability. The biochemical properties of these evolved, stand-alone TrpBs converge on those induced in the native system by allosteric activation. High-resolution crystal structures of the wild-type enzyme, an intermediate in the lineage, and the final variant, encompassing five distinct chemical states, show that activating mutations have only minor structural effects on their immediate environment. Instead, mutation stabilizes the large-scale motion of a subdomain to favor an otherwise transiently populated closed conformational state. This increase in stability enabled the first structural description of Trp covalently bound in a catalytically active TrpB, confirming key features of catalysis. These data combine to show that sophisticated models of allostery are not a prerequisite to recapitulating its complex effects via directed evolution, opening the way to engineering stand-alone versions of diverse allosteric enzymes.

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Section: Introductionmentioning

confidence: 99%

Directed Evolution Mimics Allosteric Activation by Stepwise Tuning of the Conformational Ensemble

et al. 2018

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“…The internal motions of proteins might assist as 'gate' in some systems, which controls ligand-protein association. In many enzymes or complexes, protein motions involve an allosteric communication to coordinate the function and reactions, which could be intrinsic to most enzymes [24]. Proteins are usually functions by administered of their dynamical character.…”

Section: Resultsmentioning

confidence: 99%

Combining Docking and Molecular Dynamic of Protease from Bacillus lehensis G1

Sulaiman¹

2018

JESR

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Protease is an enzyme that catalysed the hydrolysis of protein into peptide. Application of protease in industry has been linked with cost effective substrates and complex of enzyme-substrate stability. Molecular docking approach has identified casein as a preference substrates. However, lack of data on casein mode of binding to protease and enzyme stability represents a limitation for its production and structural optimization. In this study, we have used a molecular dynamic (MD) to examine the stability of complex enzyme-substrate of protease from Bacillus lehensis G1. The 3D structure of protease (BleG1_1979) was docked with substrate casein using AutoDock Vina. Structural analysis of the substrate-binding cleft revealed a binding site of casein was predominantly at the hydrophobic region of BleG1_1979. The MD of complex BleG1_1979-casein was tested with two temperatures; 298 K and 310 K using GROMACS v5.1.4. MD simulation showed a stable behaviour of BleG1_1979 over the 20 ns simulation period. The molecular docking and MD simulation suggested that the production of protease from B. lehensis G1 by utilization of casein and the stability of complex protease-casein could be a potential application to generate a cost effective enzyme to be develop for industrial use

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“…On the other hand, the study of the Ni(II) bound form of SyInrS requires the development of specific force field parameters able to model a Ni(II) ion in the square planar coordination. In recent years the improvement of the computational capabilities and the development of accelerated sampling techniques for molecular simulations [37,38] have made available the tools for the exploration of large conformational changes at the atomistic level [39][40][41]. In the case of metalloproteins, the use of accelerated sampling techniques requires the use of ad hoc designed force field parameters for the metal ions, able to reproduce structural, thermodynamic, and kinetic observables [42].…”

Section: Discussionmentioning

confidence: 99%

Molecular Modelling of the Ni(II)-Responsive Synechocystis PCC 6803 Transcriptional Regulator InrS in the Metal Bound Form

Barchi

Musiani

2019

Inorganics

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InrS (internal nickel-responsive sensor) is a transcriptional regulator found in cyanobacteria that represses the transcription of the nickel exporter NrsD in the apo form and de-represses expression of the exporter upon Ni(II) binding. Although a crystal structure of apo-InrS from Synechocystis PCC 6803 has been reported, no structure of the protein with metal ions bound is available. Here we report the results of a computational study aimed to reconstruct the metal binding site by taking advantage of recent X-ray absorption spectroscopy (XAS) data and to envisage the structural rearrangements occurring upon Ni(II) binding. The modelled Ni(II) binding site shows a square planar geometry consistent with experimental data. The structural details of the conformational changes occurring upon metal binding are also discussed in the framework of trying to rationalize the different affinity of the apo- and holo-forms of the protein for DNA.

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Investigation of Structural Dynamics of Enzymes and Protonation States of Substrates Using Computational Tools

Cited by 13 publications

References 111 publications

Directed Evolution Mimics Allosteric Activation by Stepwise Tuning of the Conformational Ensemble

Directed Evolution Mimics Allosteric Activation by Stepwise Tuning of the Conformational Ensemble

Combining Docking and Molecular Dynamic of Protease from Bacillus lehensis G1

Molecular Modelling of the Ni(II)-Responsive Synechocystis PCC 6803 Transcriptional Regulator InrS in the Metal Bound Form

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