Conformational Landscapes of Halohydrin Dehalogenases and Their Accessible Active Site Tunnels

Estévez‐Gay, Miquel; Iglésias-Fernández, Javier; Osuna, Sílvia

doi:10.3390/catal10121403

Cited by 11 publications

(12 citation statements)

References 46 publications

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“…There are, however, also subtype‐specific differences observed such as the lack of a β‐strand and an α‐helix in HheB in comparison with HheA/A2, HheC and HheG [9]. The conformational landscape of some of these representative subclasses of HHDHs has also been computationally studied by means of molecular dynamics (MD) simulations [11]. Substantial deviations in the loops located adjacent to the active site and halide binding pockets were observed among HHDH subclasses, which were found to impact the available tunnels for epoxide binding to the active site.…”

Section: Introductionmentioning

confidence: 99%

Insights into the molecular determinants of thermal stability in halohydrin dehalogenase HheD2

et al. 2021

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Halohydrin dehalogenases (HHDHs) are promising enzymes for application in biocatalysis due to their promiscuous epoxide ring-opening activity with various anionic nucleophiles. So far, seven different HHDH subtypes A to G have been reported with subtype D containing the by far largest number of enzymes. Moreover, several characterized members of subtype D have been reported to display outstanding characteristics such as high catalytic activity, broad substrate spectra or remarkable thermal stability. Yet, no structure of a D-type HHDH has been reported to date that could be used to investigate and understand those features on a molecular level. We therefore solved the crystal structure of HheD2 from gamma proteobacterium HTCC2207 at 1.6Å resolution and used it as a starting point for targeted mutagenesis in combination with molecular dynamics (MD) simulation, in order to study the low thermal stability of HheD2 in comparison with other members of subtype D. This revealed a hydrogen bond between conserved residues Q160 and D198 to be connected with a high catalytic activity of this enzyme. Moreover, a flexible surface region containing two α-helices was identified to impact thermal stability of HheD2. Exchange of this surface region by residues of HheD3 yielded a variant with 10°C higher melting temperature and reaction temperature optimum. Overall, our results provide important insights into the structure-function relationship of HheD2 and presumably for other D-type HHDHs.

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Section: Introductionmentioning

confidence: 99%

Insights into the molecular determinants of thermal stability in halohydrin dehalogenase HheD2

et al. 2021

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“…The C‐terminal domain of each subunit interacts with the diagonal subunit of another dimer. Such a C‐terminal extension is absent in other HHDHs [48] . Although it is not essential for catalytic activity, it is found to regulate HheC's enantioselectivity [49] .…”

Section: Resultsmentioning

confidence: 99%

Inhibitory Effect of DMSO on Halohydrin Dehalogenase: Experimental and Computational Insights into the Influence of an Organic Co‐solvent on the Structural and Catalytic Properties of a Biocatalyst

Milčić

Stepanić

Crnolatac

et al. 2022

Chemistry A European J

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Although the application of organic solvents in biocatalysis is well explored, in-depth understanding of the interactions of solvent with proteins, in particular oligomeric ones, is still scant. Understanding these interactions is essential in tailoring enzymes for industrially relevant catalysis in nonaqueous media. In our study, the homotetrameric enzyme halohydrin dehalogenase (HHDH) from Agrobacterium radiobacter AD1 (HheC) was investigated, as a model system, in DMSO/water solvent mixtures. DMSO, the most commonly used co-solvent for biocatalytic transformations, was found to act as a mixed-type inhibitor with a prevalent competitive contribution. Even 5 % (v/v) DMSO inhibits the activity of HheC by half. Molecular dynamics (MD) simulations showed that DMSO keeps close to Ser-Tyr catalytic residues forming alternate H-bonds with them. Stability measurements paired with differential scanning calorimetry, dynamic light scattering methods and MD studies revealed that HheC maintains its structural integrity with as much as 30 % (v/v) DMSO.

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“…The work also calls for further ITC studies on enzyme variants for fundamental studies to identify the involvement of positions of interest on reaction component binding or conversion . Finally, molecular modeling of the binding of reaction components can provide atomistic insights into binding processes. − Because our approach is amenable to automation and scale-up for high-throughput, the combination of such diverse approaches will provide the high-quality data needed for the engineering of enzymes and biocatalytic processes through machine learning to speed up the future development of industrial biocatalysis. ,− …”

Section: Discussionmentioning

confidence: 99%

Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

et al. 2021

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To apply enzymes in technical processes, a detailed understanding of the molecular mechanisms is required. Kinetic and thermodynamic parameters of enzyme catalysis are crucial to plan, model, and implement biocatalytic processes more efficiently. While the kinetic parameters, K m and k cat , are often accessible by optical methods, the determination of thermodynamic parameters requires more sophisticated methods. Isothermal titration calorimetry (ITC) allows the label-free and highly sensitive analysis of kinetic and thermodynamic parameters of individual steps in the catalytic cycle of an enzyme reaction. However, since ITC is susceptible to interferences due to denaturation or agglomeration of the enzymes, the homogeneity of the enzyme sample must always be considered, and this can be accomplished by means of dynamic light scattering (DLS) analysis. We here report on the use of an ITC-dependent work flow to determine both the kinetic and the thermodynamic data for a cofactor-dependent enzyme. Using a standardized approach with the implementation of sample quality control by DLS, we obtain high-quality data suitable for the advanced modeling of the enzyme reaction mechanism. Specifically, we investigated stereoselective reactions catalyzed by the NADPH-dependent ketoreductase Gre2p under different reaction conditions. The results revealed that this enzyme operates with an ordered sequential mechanism and is affected by substrate or product inhibition depending on the reaction buffer. Data reproducibility is ensured by specifying standard operating procedures, using programmed workflows for data analysis, and storing all data in a F.A.I.R. (findable, accessible, interoperable, and reusable) repository (https://doi.org/10.15490/fairdomhub.1.investigation.464.1). Our work highlights the utility for combined binding and kinetic studies for such complex multisubstrate reactions.

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Conformational Landscapes of Halohydrin Dehalogenases and Their Accessible Active Site Tunnels

Cited by 11 publications

References 46 publications

Insights into the molecular determinants of thermal stability in halohydrin dehalogenase HheD2

Insights into the molecular determinants of thermal stability in halohydrin dehalogenase HheD2

Inhibitory Effect of DMSO on Halohydrin Dehalogenase: Experimental and Computational Insights into the Influence of an Organic Co‐solvent on the Structural and Catalytic Properties of a Biocatalyst

Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

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