Different Structural Origins of the Enantioselectivity of Haloalkane Dehalogenases toward Linear β‐Haloalkanes: Open–Solvated versus Occluded–Desolvated Active Sites

Lišková, Veronika; Štěpánková, Veronika; Bednář, David; Brezovský, Jan; Prokop, Zbyněk; Chaloupková, Radka; Damborský, Jiřı́

doi:10.1002/anie.201611193

Cited by 15 publications

(6 citation statements)

References 30 publications

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“…The mutant recombinant genes of dhaA04 (C176Y), dhaA31 (I135F, C176Y, V245F, L246I, and Y273F), dhaA137 (L246I), and dhaA138 (V245F) were constructed using techniques of directed evolution and site-directed mutagenesis as described previously. The recombinant gene of dhaA133 (C176Y, V245F) was synthesized artificially (GeneArt, LifeTechnologies, Germany) according to the wild-type sequence, and the gene sequence (Figure S2) was optimized for an expression in Escherichia coli .…”

Section: Methodssupporting

confidence: 78%

Catalytic Cycle of Haloalkane Dehalogenases Toward Unnatural Substrates Explored by Computational Modeling

Marques

Dunajova

Prokop

et al. 2017

J. Chem. Inf. Model.

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The anthropogenic toxic compound 1,2,3-trichloropropane is poorly degradable by natural enzymes. We have previously constructed the haloalkane dehalogenase DhaA31 by focused directed evolution ( Pavlova, M. et al. Nat. Chem. Biol. 2009 , 5 , 727 - 733 ), which is 32 times more active than the wild-type enzyme and is currently the most active variant known against that substrate. Recent evidence has shown that the structural basis responsible for the higher activity of DhaA31 was poorly understood. Here we have undertaken a comprehensive computational study of the main steps involved in the biocatalytic hydrolysis of 1,2,3-trichloropropane to decipher the structural basis for such enhancements. Using molecular dynamics and quantum mechanics approaches we have surveyed (i) the substrate binding, (ii) the formation of the reactive complex, (iii) the chemical step, and (iv) the release of the products. We showed that the binding of the substrate and its transport through the molecular tunnel to the active site is a relatively fast process. The cleavage of the carbon-halogen bond was previously identified as the rate-limiting step in the wild-type. Here we demonstrate that this step was enhanced in DhaA31 due to a significantly higher number of reactive configurations of the substrate and a decrease of the energy barrier to the S2 reaction. C176Y and V245F were identified as the key mutations responsible for most of those improvements. The release of the alcohol product was found to be the rate-limiting step in DhaA31 primarily due to the C176Y mutation. Mutational dissection of DhaA31 and kinetic analysis of the intermediate mutants confirmed the theoretical observations. Overall, our comprehensive computational approach has unveiled mechanistic details of the catalytic cycle which will enable a balanced design of more efficient enzymes. This approach is applicable to deepen the biochemical knowledge of a large number of other systems and may contribute to robust strategies in the development of new biocatalysts.

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Section: Methodssupporting

confidence: 78%

Catalytic Cycle of Haloalkane Dehalogenases Toward Unnatural Substrates Explored by Computational Modeling

Marques

Dunajova

Prokop

et al. 2017

J. Chem. Inf. Model.

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“…In most enzymes (>60%), the active site is located in a deep internal cavity connected to the bulk solvent by channels or tunnels . Engineering enzyme access tunnels has become an effective strategy for directed protein evolution, as it can substantially increase enzyme activity, specificity, promiscuity, enantioselectivity, and stability. − Specifically, epoxide-hydrolase activity was enhanced 42-fold by mutating large amino acid residues (Leu, Met, and Phe) into smaller ones (Ala) in the substrate tunnel; this change was accompanied by significantly increased product-release rate and the transfer of substrate specificity to bulky epoxide . Membrane-bound fatty aldehyde dehydrogenase uses a complex gated tunnel to control the substrate entering its active site; removal of this tunnel did not affect overall catalytic activity but changed its specificity toward short-chain substrates .…”

Section: Introductionmentioning

confidence: 99%

Open Gate of Corynebacterium glutamicum Threonine Deaminase for Efficient Synthesis of Bulky α-Keto Acids

Song

Gao

et al. 2020

ACS Catal.

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Threonine deaminase (TD, EC 4.3.1.19) mediates α,βelimination (deamination), which has untapped potential in the synthesis of unnatural α-keto acids. However, the narrow substrate scope of wild-type TD limits its application. This issue could be overcome by engineering the substrate tunnel of the enzyme. Here, Corynebacterium glutamicum TD (CgTD) was used as a model, and its substrate tunnel was identified as a gating element. On the basis of the gating characteristics and constitution of the substrate access tunnel of CgTD, an "open-gate" strategy was developed to improve its substrate scope toward bulky substrates. The best variant obtained following this approach, CgTD Mu7 , exhibited 90.6-fold greater catalytic efficiency (k cat / K M ) toward a bulky substrate such as phenylserine. The improvement was due to more efficient substrate coupling, deprotonation, and imine hydrolysis. CgTD Mu7 was used for efficient production of both natural and unnatural α-keto acids, including 2-oxobutyric acid (83.4 g L −1 , 99% conversion), phenylpyruvic acid (72.5 g L −1 , 95% conversion), 2-oxovaleric acid (21.1 g L −1 , 91% conversion), and 2-oxo-4-phenylbutyric acid (29.9 g L −1 , 84% conversion). The proposed strategy provides a theoretical basis for the synthesis of α-keto acids and is also effective for engineering other similar enzymes.

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“…To reduce the screening effort, the structure-guided rational or semi-rational design strategy is highly promising . It generally relies on clear structural knowledge of the enzymes and theoretical simulations to reshape the substrate binding cavity or entrance tunnel. − For amidase, several groups have used protein engineering to improve enzymatic stability or efficiency, − but there is no report on manipulation of chiral transformation, especially enantioselectivity.…”

Section: Introductionmentioning

confidence: 99%

Reversal and Amplification of the Enantioselectivity of Biocatalytic Desymmetrization toward Meso Heterocyclic Dicarboxamides Enabled by Rational Engineering of Amidase

Zhao

et al. 2021

ACS Catal.

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By rational engineering of amidase, the efficient biocatalytic desymmetrization of meso O-heterocyclic dicarboxamides for synthesizing both antipodes of functionalized cyclic motifs was presented. Based on the enzyme-substrate binding model suggested by molecular docking, a rational mutagenesis strategy was established. The reversal and amplification of enantioselectivity of amidase were achieved by generating and testing only 10 variants. This enabled the quick access of both antipodes of products in very good yields and up to 99.5% ee under mild conditions. The engineered biocatalyst exhibits a wide substrate promiscuity and can expand to both N-heterocyclic and carbocyclic dicarboxamides with the retained high efficiency and excellent enantioselectivity. The desymmetrization mechanism for amidase, including the wild-type and the variant, was revealed by molecular dynamics simulations and quantum mechanical/molecular mechanical modeling. It suggests a delicate cooperation between the activation and binding sites by nesting the cyclic skeleton of the substrates.

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Different Structural Origins of the Enantioselectivity of Haloalkane Dehalogenases toward Linear β‐Haloalkanes: Open–Solvated versus Occluded–Desolvated Active Sites

Cited by 15 publications

References 30 publications

Catalytic Cycle of Haloalkane Dehalogenases Toward Unnatural Substrates Explored by Computational Modeling

Catalytic Cycle of Haloalkane Dehalogenases Toward Unnatural Substrates Explored by Computational Modeling

Open Gate of Corynebacterium glutamicum Threonine Deaminase for Efficient Synthesis of Bulky α-Keto Acids

Reversal and Amplification of the Enantioselectivity of Biocatalytic Desymmetrization toward Meso Heterocyclic Dicarboxamides Enabled by Rational Engineering of Amidase

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