Active-Site Flexibility and Substrate Specificity in a Bacterial Virulence Factor: Crystallographic Snapshots of an Epoxide Hydrolase

Hvorecny, Kelli L.; Bahl, Christopher D.; Kitamura, Seiya; Lee, Kin Sing Stephen; Morisseau, Christophe; Madden, Dean R.

doi:10.1016/j.str.2017.03.002

Cited by 17 publications

(19 citation statements)

References 57 publications

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“…In this study, decreased volume of stereo-selective pocket correlates with increased stereo-selectivity. Enzyme selectivity also depends on the active site flexibility, wherein a less flexible site is more selective [66]. All these results are in line with our rational approach which results in DM-GTL with excellent fatty acid selectivity.…”

Section: Discussionsupporting

confidence: 86%

Substrate structure and computation guided engineering of a Lipase for Omega-3 fatty acid selectivity

Moharana

Rao

2019

Preprint

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AbstractOptimum health benefits of omega-3 fatty acids (ω-3 FAs) require it to be concentrated in its natural sources. Fatty acid selectivity of lipase governs the efficacy of the production of lipase-mediated ω-3 FAs concentrates. We attempted to improve the fatty acid selectivity of a lipase from thermophilic bacteriumGeobacillus thermoleovorans(GTL) by two approaches. In a semi-rational approach, six amino acid positions of GTL interacting with the substrate, were identified by docking and were subjected to site-saturation mutagenesis. Three best substitutions were incorporated into GTL(CM-GTL). Hydrolysis of oil by lipase was monitored in a pH-Stat and the fatty acids released at various time points were analyzed by GC-MS.CM-GTL showed a significant improvement in discrimination against DHA during hydrolysis. In the second approach based on rational design, the active site was narrowed by incorporating heavier amino acids in the lining of acyl-binding pocket to hinder access to bulky ω-3 FAs. For this purpose, two amino acids surrounding the opening of the acyl pocket were replaced with the next heavier amino acids and the affinities were evaluatedin silico.The double mutant, thus deigned, was found to be excellent in discriminating the ω-3 FAs during hydrolysis of triglycerides. Engineering the binding pocket of a complex substrate, such as a triglyceride, with the supportive information on substrate structure and its binding modes with the enzyme provided by computational methods, has resulted in designing two efficient lipase variants with improved substrate selectivity.

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Section: Discussionsupporting

confidence: 86%

Substrate structure and computation guided engineering of a Lipase for Omega-3 fatty acid selectivity

Moharana

Rao

2019

Preprint

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“…In this study, decreased volume of stereo-selective pocket correlates with increased stereo-selectivity. Enzyme selectivity also depends on the active site flexibility, wherein a less flexible site is more selective [67]. All these results are in line with our rational approach in designing the DM-GTL.…”

Section: Discussionsupporting

confidence: 84%

Substrate structure and computation guided engineering of a lipase for omega-3 fatty acid selectivity

Moharana

Rao

2020

PLoS ONE

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Enrichment of omega-3 fatty acids (ω-3 FAs) in natural oils is important to realize their health benefits. Lipases are promising catalysts to perform this enrichment, however, fatty acid specificity of lipases is poor. We attempted to improve the fatty acid selectivity of a lipase from Geobacillus thermoleovorans (GTL) by two approaches. In a semi-rational approach, amino acid positions critical for binding were identified by docking the substrate to the GTL and best substitutes at these positions were identified by site saturation mutagenesis followed by screening to obtain a variant of GTL (CM-GTL). In the second approach based on rational design, a variant of GTL was designed (DM-GTL) wherein the active site was narrowed by incorporating two heavier amino acids in the lining of acyl-binding pocket to hinder access to bulky ω-3 FAs. The affinities DM-GTL with designed substrates were evaluated in silico. Both, CM-GTL and DM-GTL have shown excellent ability to discriminate against the ω-3 FAs during hydrolysis of oils. Engineering the binding pocket of an enzyme of a complex substrate, such as a triglyceride, by incorporating the information on substrate structure and computationally derived binding modes, has resulted in designing two efficient lipase variants with improved substrate selectivity.

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“…This proposed critera is based, in part, on the concept of 'near attack conformers' [266,267], which is used as a predictor of the enantioselective potential of mutants in a number of studies [268,269,46,47]. The criteria used by Reetz et al [252], however, does not account of the possibility of mixed or alternative regioselectivity that may occur in EH reactions [29] (discussed further in Chapter 7). Here a more comprehensive set of distance criteria are applied, specifically optimal poses for both enantiomers are ranked by the distances between Asp192Oδ 1:C-1, Asp192Oδ 1:C-2, Asp192Oδ 2:C-1 and Asp192Oδ 2:C-2.…”

Section: Analysis Of Docking Resultsmentioning

confidence: 99%

“…The mean distances between Asp192Oδ 1 and Oδ 2 to both C-1 and C-2 are similar for both enantiomers, suggesting the possibility of a lack of regioselectivity. A lack of regioselectivity is supported by a recent study where ester intermediates were covalently linked to the EH from Pseudomonas aeruginosa and successfully trapped and crystallised [29]. The study demonstrated that the enzyme could potentially attack a distinct carbon atom on the epoxide moiety of each enantiomeric pair of the substrate.…”

Section: Discussionmentioning

confidence: 99%

“…A wealth of structural and biochemical data has been generated for EHs [1,[16][17][18][19][20][21][22][23], however the origin(s) of their enantio-and regioselective (Figure 1.1) properties remains uncertain [24]. EHs therefore continue to be actively studied in order to better understand, predict and improve their selectivities [24][25][26][27][28][29][30][31][32]. This thesis contributes to this endeavour by developing and exploring different, yet complementary, computational methods for the modelling of the enantioselectivity of an EH from Aspergillus niger (AnEH).…”

Section: General Introductionmentioning

confidence: 99%

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Computational modelling of enzymes: predicting and understanding the selectivity of an epoxide hydrolase

Zaugg¹

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Enzymes provide improved catalytic efficiency, renewability and higher selectivity compared to traditional chemical synthesis methods. Stereoselective enzymes in particular are highly desired within industry for the production of fine chemicals and drugs. Epoxide hydrolases (EH) are a family of enzymes whose selective properties facilitate the production of valuable intermediates used for the synthesis of many pharmaceuticals. There is a wealth of structural and biochemical data available for EHs, however the origin of their selectivity remains uncertain. Computational and statistical methods can aid in the design and discovery of selective enzymes and interpretation of the experimental data produced during their characterisation. This thesis focuses on the EH from the fungus Aspergillus niger (AnEH) and its aim is two-fold. The first aim is to explore and develop machine learning methods to predict selectivity enhancing mutations for AnEH. The second aim is to get insight into the mechanistic determinants of AnEH selectivity through molecular modelling methods. Publications during candidatureBook chapters • Zaugg J., Gumulya Y., Gillam E. M. J. and Bodén M. (2014) Computational tools for directed evolution: a comparison of prospective and retrospective strategies. Methods in Molecular Biology 1179:315-333 Contributions by others to the thesis Chapter 2 Zaugg J (candidate) and Bodén M researched and wrote the manuscript. Zaugg J, Bodén M, Gumulya Y and Gillam E edited the manuscript. Chapter 3 Zaugg J (candidate) carried out all computational experiments. Gumulya Y provided experimental data. Bodén M and Malde AK provided guidance in experimental design and analysis of results. Chapter 4 Zaugg J (candidate) carried out all computational experiments. Gumulya Y provided experimental data. Bodén M provided guidance in experimental design and analysis of results. Bodén M wrote much of the software (Bnkit) used for creating and training Bayesian networks. Wang Y developed code to allow specification of Dirichlet priors for Bayesian networks. Essebier A provided code for testing Bayesian networks. Chapter 6 Zaugg J (candidate) designed and carried out all computational experiments. Gumulya Y provided experimental data. Chapter 7 Zaugg J (candidate) carried out all computational experiments and designed the protocol used for docking of ligands. Malde AK and Mark AE provided guidance in the design and implementation of molecular dynamics simulations and free energy calculations and the analysis of results. Gumulya Y provided experimental data. Zaugg J, Gumulya Y, Bodén M, Mark AE and Malde AK wrote and edited the manuscript. IX Statement of parts of the thesis submitted to qualify for the award of another degree None. X Research Involving Human or Animal Subjects No animal or human subjects were involved in this research. XI Abstract I List of Figures XVI List of Tables XIX

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Active-Site Flexibility and Substrate Specificity in a Bacterial Virulence Factor: Crystallographic Snapshots of an Epoxide Hydrolase

Cited by 17 publications

References 57 publications

Substrate structure and computation guided engineering of a Lipase for Omega-3 fatty acid selectivity

Substrate structure and computation guided engineering of a Lipase for Omega-3 fatty acid selectivity

Substrate structure and computation guided engineering of a lipase for omega-3 fatty acid selectivity

Computational modelling of enzymes: predicting and understanding the selectivity of an epoxide hydrolase

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