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Structural and catalytic effects of surface loop-helix transplantation within haloalkane dehalogenase family
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SYSNO ASEP 0539596 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Structural and catalytic effects of surface loop-helix transplantation within haloalkane dehalogenase family Author(s) Marek, M. (CZ)
Chaloupková, R. (CZ)
Prudnikova, T. (CZ)
Sato, Y. (JP)
Řezáčová, Pavlína (UMG-J) RID
Nagata, Y. (JP)
Smatanová, I.K. (CZ)
Damborský, J. (CZ)Number of authors 8 Source Title Computational and Structural Biotechnology Journal. - : Elsevier - ISSN 2001-0370
Roč. 18, June (2020), s. 1352-1362Number of pages 11 s. Publication form Online - E Language eng - English Country SE - Sweden Keywords Haloalkane dehalogenase (HLD) ; Biocatalysis ; Loop-helix transplantation ; X-ray crystallography ; Enantioselectivity ; Access tunnel ; Enzyme engineering ; Protein design Subject RIV EB - Genetics ; Molecular Biology OECD category Biochemistry and molecular biology Method of publishing Open access Institutional support UMG-J - RVO:68378050 UT WOS 000607350300011 DOI 10.1016/j.csbj.2020.05.019 Annotation Engineering enzyme catalytic properties is important for basic research as well as for biotechnological applications. We have previously shown that the reshaping of enzyme access tunnels via the deletion of a short surface loop element may yield a haloalkane dehalogenase variant with markedly modified substrate specificity and enantioselectivity. Here, we conversely probed the effects of surface loop-helix transplantation from one enzyme to another within the enzyme family of haloalkane dehalogenases. Precisely, we transplanted a nine-residue long extension of L9 loop and alpha 4 helix from DbjA into the corresponding site of DbeA. Biophysical characterization showed that this fragment transplantation did not affect the overall protein fold or oligomeric state, but lowered protein stability (Delta T-m =5 to 6 degrees C). Interestingly, the crystal structure of DbeA mutant revealed the unique structural features of enzyme access tunnels, which are known determinants of catalytic properties for this enzyme family. Biochemical data confirmed that insertion increased activity of DbeA with various halogenated substrates and altered its enantioselectivity with several linear beta-bromoalkanes. Our findings support a protein engineering strategy employing surface loop-helix transplantation for construction of novel protein catalysts with modified catalytic properties. (C) 2020 The Author(s). Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology. Workplace Institute of Molecular Genetics Contact Nikol Škňouřilová, nikol.sknourilova@img.cas.cz, Tel.: 241 063 217 Year of Publishing 2021 Electronic address https://www.sciencedirect.com/science/article/pii/S2001037020302828?via%3Dihub
Number of the records: 1