Pathways and Mechanisms for Product Release in the Engineered Haloalkane Dehalogenases Explored Using Classical and Random Acceleration Molecular Dynamics Simulations

Klvaňa, Martin; Pavlová, Martina; Koudeláková, Táňa; Chaloupková, Radka; Dvořák, Pavel; Prokop, Zbyněk; Stsiapanava, A.; Kutý, Michal; Smatanová, Ivana Kutá; Dohnálek, Jan; Kulhánek, Petr; Wade, Rebecca C.; Damborský, Jiřı́

doi:10.1016/j.jmb.2009.06.076

Cited by 96 publications

(100 citation statements)

References 93 publications

(171 reference statements)

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“…In most cases, the product release and substrate access share the same tunnel in proteins (31)(32)(33). However, the product-release tunnel in enzymes such as haloalkane dehalogenases is distinct from the substrate access tunnel, which sometimes makes the product release step a rate-limiting bottleneck, thereby providing a new target for efficient protein engineering (34)(35)(36). Previously, there was no report of an independent product-release site in EHs.…”

Section: Discussionmentioning

confidence: 99%

Engineering of an epoxide hydrolase for efficient bioresolution of bulky pharmaco substrates

Kong

Yuan

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Optically pure epoxides are essential chiral precursors for the production of (S)-propranolol, (S)-alprenolol, and other β-adrenergic receptor blocking drugs. Although the enzymatic production of these bulky epoxides has proven difficult, here we report a method to effectively improve the activity of BmEH, an epoxide hydrolase from Bacillus megaterium ECU1001 toward α-naphthyl glycidyl ether, the precursor of (S)-propranolol, by eliminating the steric hindrance near the potential product-release site. Using X-ray crystallography, mass spectrum, and molecular dynamics calculations, we have identified an active tunnel for substrate access and product release of this enzyme. The crystal structures revealed that there is an independent product-release site in BmEH that was not included in other reported epoxide hydrolase structures. By alanine scanning, two mutants, F128A and M145A, targeted to expand the potential product-release site displayed 42 and 25 times higher activities toward α-naphthyl glycidyl ether than the wild-type enzyme, respectively. These results show great promise for structure-based rational design in improving the catalytic efficiency of industrial enzymes for bulky substrates.epoxide hydrolase | X-ray crystallography | protein engineering | product release | bulky substrate O ptically pure epoxides and the corresponding vicinal diols are valuable chiral building blocks for the production of pharmaceutically active compounds and other fine chemicals (1). Existing approaches for preparing enantiopure epoxides and diols include the asymmetric epoxidation or dihydroxylation of olefin substrates and the resolution of racemic epoxides. These reactions can be accomplished with either chemical catalysts such as chiral salen cobalt complexes and porphyrin manganese adducts or biocatalysts such as monooxygenases and epoxide hydrolases (EHs) (2-4). In the past two decades, EHs have received much attention because they are cofactor-independent enzymes that are "easy to use" for catalyzing the hydrolysis of racemic epoxides to yield highly enantiopure epoxides and vicinal diols (1, 5, 6). However, application of EHs in laboratory and industry was often hindered by their narrow substrate scope, low enantioselectivity, and regioselectivity, or product inhibition (7,8).Many protein-engineering efforts have been made to overcome these drawbacks (9, 10). For example, directed evolution by error-prone PCR or DNA shuffling has been used to enhance the activity and enantioselectivity of EHs (11-13). Structureguided mutagenesis also generated a few EH variants with improved catalytic performance (14-16). The strategy of iterative Combinatorial Active Site-Saturation Test (CAST) combines the rational approach and directed evolution to yield high-quality and small focused mutant libraries for screening EHs with better enantioselectivity (7,17). By mutating residues at the substratebinding site, the substrates of EHs have been expanded to include cyclic meso-epoxides, phenyl glycidyl ether (PGE) derivatives, and other...

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Section: Discussionmentioning

confidence: 99%

Engineering of an epoxide hydrolase for efficient bioresolution of bulky pharmaco substrates

Kong

Yuan

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Several experimental and computational studies of enzymes with buried active sites, such as acetylcholinesterases (6,44), haloalkane dehalogenases (12,43), cytochromes P450 (7,18,45,46), and lipases (47)(48)(49)(50) have been performed and have underlined the importance of enzyme tunnels for catalytic activity, specificity, and enantioselectivity. Here, we report the first combined experimental and computational study of the effect of a tunnel-lining mutation on the mechanism of the biotechnologically interesting enzyme, haloalkane dehalogenase LinB.…”

Section: Discussionmentioning

confidence: 99%

A Single Mutation in a Tunnel to the Active Site Changes the Mechanism and Kinetics of Product Release in Haloalkane Dehalogenase LinB

Biedermannová

Prokop

Góra

et al. 2012

Journal of Biological Chemistry

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Background: Tunnel properties affect ligand passage in enzymes with buried active sites. Results: A tunnel mutation from leucine to tryptophan changes the mechanism of bromide ion release from haloalkane dehalogenase LinB. Conclusion:Interactions of the bromide ion with the tryptophan increase free energy barrier for its passage, causing the reaction mechanism change. Significance: The results provide guidelines for enzyme engineering.

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“…Considering that ligand binding is a reversible process, this method can be used to explore substrate dissociation and binding pathways as well (19). The RAMD method has been successfully employed to study ligand dissociation paths in cytochrome P450 (19), rhodopsin (22), haloalkane dehalogenase (23), histone deacetylase (24), heme oxygenase (25), thyroid hormone receptor (26), P-glycoprotein (27), and B-RAF kinase (28), among many others.…”

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confidence: 99%

A Hidden Active Site in the Potential Drug Target Mycobacterium tuberculosis dUTPase Is Accessible through Small Amplitude Protein Conformational Changes

Lopata

Leveles

Bendes

et al. 2016

Journal of Biological Chemistry

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Edited by Norma AllewelldUTPases catalyze the hydrolysis of dUTP into dUMP and pyrophosphate to maintain the proper nucleotide pool for DNA metabolism. Recent evidence suggests that dUTPases may also represent a selective drug target in mycobacteria because of the crucial role of these enzymes in maintaining DNA integrity. Nucleotide-hydrolyzing enzymes typically harbor a buried ligand-binding pocket at interdomain or intersubunit clefts, facilitating proper solvent shielding for the catalyzed reaction. The mechanism by which substrate binds this hidden pocket and product is released in dUTPases is unresolved because of conflicting crystallographic and spectroscopic data. We sought to resolve this conflict by using a combination of random acceleration molecular dynamics (RAMD) methodology and structural and biochemical methods to study the dUTPase from Mycobacterium tuberculosis. In particular, the RAMD approach used in this study provided invaluable insights into the nucleotide dissociation process that reconciles all previous experimental observations. Specifically, our data suggest that nucleotide binding takes place as a small stretch of amino acids transiently slides away and partially uncovers the active site. The in silico data further revealed a new dUTPase conformation on the pathway to a relatively open active site. To probe this model, we developed the Trp 21 reporter and collected crystallographic, spectroscopic, and kinetic data that confirmed the interaction of Trp 21 with the active site shielding C-terminal arm, suggesting that the RAMD method is effective. In summary, our computational simulations and spectroscopic results support the idea that small loop movements in dUTPase allow the shuttling of the nucleotides between the binding pocket and the solvent.

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Pathways and Mechanisms for Product Release in the Engineered Haloalkane Dehalogenases Explored Using Classical and Random Acceleration Molecular Dynamics Simulations

Cited by 96 publications

References 93 publications

Engineering of an epoxide hydrolase for efficient bioresolution of bulky pharmaco substrates

Engineering of an epoxide hydrolase for efficient bioresolution of bulky pharmaco substrates

A Single Mutation in a Tunnel to the Active Site Changes the Mechanism and Kinetics of Product Release in Haloalkane Dehalogenase LinB

A Hidden Active Site in the Potential Drug Target Mycobacterium tuberculosis dUTPase Is Accessible through Small Amplitude Protein Conformational Changes

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