Advanced database mining of efficient haloalkane dehalogenases by sequence and structure bioinformatics and microfluidics

Vasina, Michal; Vaňáček, Pavel; Hon, Jiří; Kovar, David R.; Faldynova, Hana; Kunka, Antonín; Buryška, Tomáš; Badenhorst, Christoffel P. S.; Mazurenko, Stanislav; Bednář, David; Stavrakis, Stavros; Bornscheuer, Uwe T.; deMello, Andrew J.; Damborský, Jiřı́; Prokop, Zbyněk

doi:10.1016/j.checat.2022.09.011

Cited by 21 publications

(24 citation statements)

References 64 publications

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“…DhmeA originates from the extremophilic archaeon Haloferax mediterranei that inhabits hypersaline water ecosystems, such as saltwater evaporation ponds (Han et al, 2012;Oren & Hallsworth, 2014). Previous experimental characterizations confirmed many of the commonly observed features of the HLD-III family: low-expression yield and poor solubility, oligomerization tendency and polydisperse heterogeneity of the final product, as well as low dehalogenase activity (Vanacek et al, 2018;Vasina et al, 2022). One of its unique features is the melting temperature of 71 C, currently making it the most natively thermostable wild-type HLD (Vanacek et al, 2018).…”

Section: Introductionmentioning

confidence: 83%

Multimeric structure of a subfamily III haloalkane dehalogenase‐like enzyme solved by combination of cryo‐EM and x‐ray crystallography

et al. 2023

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Haloalkane dehalogenase (HLD) enzymes employ an SN2 nucleophilic substitution mechanism to erase halogen substituents in diverse organohalogen compounds. Subfamily I and II HLDs are well‐characterized enzymes, but a mode and purpose of multimerization of subfamily III HLDs are unknown. Here we probe the structural organization of DhmeA, a subfamily III HLD‐like enzyme from the archaeon Haloferax mediterranei, by combining cryo‐electron microscopy (cryo‐EM) and X‐ray crystallography. We show that full‐length wild‐type DhmeA forms diverse quaternary structures, ranging from small oligomers to large supramolecular ring‐like assemblies of various sizes and symmetries. We optimized sample preparation steps, enabling three‐dimensional reconstructions of an oligomeric species by single‐particle cryo‐EM. Moreover, we engineered a crystallizable mutant (DhmeAΔGG) that provided diffraction‐quality crystals. The 3.3 Å crystal structure reveals that DhmeAΔGG forms a ring‐like 20‐mer structure with outer and inner diameter of ~200 Å and ~80 Å, respectively. An enzyme homodimer represents a basic repeating building unit of the crystallographic ring. Three assembly interfaces (dimerization, tetramerization and multimerization) were identified to form the supramolecular ring that displays a negatively charged exterior, while its interior part harboring catalytic sites is positively charged. Localization and exposure of catalytic machineries suggest a possible processing of large negatively charged macromolecular substrates.This article is protected by copyright. All rights reserved.

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Section: Introductionmentioning

confidence: 83%

Multimeric structure of a subfamily III haloalkane dehalogenase‐like enzyme solved by combination of cryo‐EM and x‐ray crystallography

et al. 2023

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“…The mutations globally increased the turnover by decreasing the activation enthalpy of the chemical reaction for the substrate 1,2-dibromoethane. The maximal catalytic efficiencies of the best variants, adjusted for substrate inhibition, are three to four times higher than DhaA115 and comparable to the most active naturally occurring or computationally designed dehalogenases to date …”

Section: Discussionmentioning

confidence: 99%

Advancing Enzyme’s Stability and Catalytic Efficiency through Synergy of Force-Field Calculations, Evolutionary Analysis, and Machine Learning

Kunka,

Marques,

Havlasek

et al. 2023

ACS Catal.

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Thermostability is an essential requirement for the use of enzymes in the bioindustry. Here, we compare different protein stabilization strategies using a challenging target, a stable haloalkane dehalogenase DhaA115. We observe better performance of automated stabilization platforms FireProt and PROSS in designing multiple-point mutations over the introduction of disulfide bonds and strengthening the intra- and the inter-domain contacts by in silico saturation mutagenesis. We reveal that the performance of automated stabilization platforms was still compromised due to the introduction of some destabilizing mutations. Notably, we show that their prediction accuracy can be improved by applying manual curation or machine learning for the removal of potentially destabilizing mutations, yielding highly stable haloalkane dehalogenases with enhanced catalytic properties. A comparison of crystallographic structures revealed that current stabilization rounds were not accompanied by large backbone re-arrangements previously observed during the engineering stability of DhaA115. Stabilization was achieved by improving local contacts including protein–water interactions. Our study provides guidance for further improvement of automated structure-based computational tools for protein stabilization.

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“…Sequence-based screening requires accurate in silico tools with low computational demands to provide a fast, cost-effective, and reliable high-throughput screening protocol to predict whether an enzyme, among several thousand candidates, is able to efficiently degrade the target polyester. An in silico protocol was proposed by Vasina et al to predict a “small-but-smart” set of efficient biocatalysts for other classes of enzymes [ 231 ]. A factor that massively affects the predictive accuracy of these tools is the capacity to disentangle the molecular mechanisms that allow for superior polyester degradation.…”

Section: Perspectivesmentioning

confidence: 99%

Microbial Enzyme Biotechnology to Reach Plastic Waste Circularity: Current Status, Problems and Perspectives

Orlando

Molla

Castellani

et al. 2023

IJMS

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The accumulation of synthetic plastic waste in the environment has become a global concern. Microbial enzymes (purified or as whole-cell biocatalysts) represent emerging biotechnological tools for waste circularity; they can depolymerize materials into reusable building blocks, but their contribution must be considered within the context of present waste management practices. This review reports on the prospective of biotechnological tools for plastic bio-recycling within the framework of plastic waste management in Europe. Available biotechnology tools can support polyethylene terephthalate (PET) recycling. However, PET represents only ≈7% of unrecycled plastic waste. Polyurethanes, the principal unrecycled waste fraction, together with other thermosets and more recalcitrant thermoplastics (e.g., polyolefins) are the next plausible target for enzyme-based depolymerization, even if this process is currently effective only on ideal polyester-based polymers. To extend the contribution of biotechnology to plastic circularity, optimization of collection and sorting systems should be considered to feed chemoenzymatic technologies for the treatment of more recalcitrant and mixed polymers. In addition, new bio-based technologies with a lower environmental impact in comparison with the present approaches should be developed to depolymerize (available or new) plastic materials, that should be designed for the required durability and for being susceptible to the action of enzymes.

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Advanced database mining of efficient haloalkane dehalogenases by sequence and structure bioinformatics and microfluidics

Cited by 21 publications

References 64 publications

Multimeric structure of a subfamily III haloalkane dehalogenase‐like enzyme solved by combination of cryo‐EM and x‐ray crystallography

Multimeric structure of a subfamily III haloalkane dehalogenase‐like enzyme solved by combination of cryo‐EM and x‐ray crystallography

Advancing Enzyme’s Stability and Catalytic Efficiency through Synergy of Force-Field Calculations, Evolutionary Analysis, and Machine Learning

Microbial Enzyme Biotechnology to Reach Plastic Waste Circularity: Current Status, Problems and Perspectives

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