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Understanding desaturation/hydroxylation activity of castor stearoyl Δ9-Desaturase through rational mutagenesis
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SYSNO ASEP 0556929 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Understanding desaturation/hydroxylation activity of castor stearoyl Δ9-Desaturase through rational mutagenesis Author(s) Tupec, Michal (UOCHB-X) RID, ORCID
Culka, Martin (UOCHB-X) ORCID
Machara, Aleš (UOCHB-X) ORCID
Macháček, Stanislav (UOCHB-X)
Bím, Daniel (UOCHB-X) ORCID, RID
Svatoš, Aleš (UOCHB-X) ORCID, RID
Rulíšek, Lubomír (UOCHB-X) RID, ORCID
Pichová, Iva (UOCHB-X) RID, ORCIDSource Title Computational and Structural Biotechnology Journal. - : Elsevier - ISSN 2001-0370
Roč. 20, March (2022), s. 1378-1388Number of pages 11 s. Language eng - English Country SE - Sweden Keywords Δ9 desaturase ; methane monooxygenase ; reaction mechanism ; proton transfer ; desaturation ; hydroxylation OECD category Biochemistry and molecular biology R&D Projects LTAUSA19148 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) LM2015064 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) EF16_019/0000729 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) Method of publishing Open access Institutional support UOCHB-X - RVO:61388963 UT WOS 000791774200011 EID SCOPUS 85126578344 DOI 10.1016/j.csbj.2022.03.010 Annotation A recently proposed reaction mechanism of soluble Δ9 desaturase (Δ9D) allowed us to identify auxiliary residues His203, Asp101, Thr206 and Cys222 localized near the di-iron active site that are supposedly involved in the proton transfer (PT) to and from the active site. The PT, along with the electron transfer (ET), seems to be crucial for efficient desaturation. Thus, perturbing the major PT chains is expected to impair the native reaction and (potentially) amplify minor reaction channels, such as the substrate hydroxylation. To verify this hypothesis, we mutated the four residues mentioned above into their counterparts present in a soluble methane monooxygenase (sMMO), and determined the reaction products of mutants. We found that the mutations significantly promote residual monohydroxylation activities on stearoyl-CoA, often at the expense of native desaturation activity. The favored hydroxylation positions are C9, followed by C10 and C11. Reactions with unsaturated substrate, oleoyl-CoA, yield erythro-9,10-diol, cis-9,10-epoxide and a mixture of allylic alcohols. Additionally, using 9- and 11-hydroxystearoyl-CoA, we showed that the desaturation reaction can proceed only with the hydroxyl group at position C11, whereas the hydroxylation reaction is possible in both cases, i.e. with hydroxyl at position C9 or C11. Despite the fact that the overall outcome of hydroxylation is rather modest and that it is mostly the desaturation/hydroxylation ratio that is affected, our results broaden understanding of the origin of chemo- and stereoselectivity of the Δ9D and provide further insight into the catalytic action of the NHFe2 enzymes. Workplace Institute of Organic Chemistry and Biochemistry Contact asep@uochb.cas.cz ; Kateřina Šperková, Tel.: 232 002 584 ; Jana Procházková, Tel.: 220 183 418 Year of Publishing 2023 Electronic address https://doi.org/10.1016/j.csbj.2022.03.010
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