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Mutations Increasing Cofactor Affinity, Improve Stability and Activity of a Baeyer-Villiger Monooxygenase.

التفاصيل البيبلوغرافية
العنوان: Mutations Increasing Cofactor Affinity, Improve Stability and Activity of a Baeyer-Villiger Monooxygenase.
المؤلفون: Mansouri HR; Institute of Applied Synthetic Chemistry, TU Wien, Getreidemarkt 9, 1060 Vienna, Austria., Gracia Carmona O; Institute of Molecular Modeling and Simulation, University of Natural Resources and Life Sciences, 1190 Vienna, Austria., Jodlbauer J; Institute of Applied Synthetic Chemistry, TU Wien, Getreidemarkt 9, 1060 Vienna, Austria., Schweiger L; Biocatalysis and Biosensing Laboratory, Department of Food Science and Technology, BOKU-University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190 Vienna, Austria., Fink MJ; Institute of Applied Synthetic Chemistry, TU Wien, Getreidemarkt 9, 1060 Vienna, Austria., Breslmayr E; Biocatalysis and Biosensing Laboratory, Department of Food Science and Technology, BOKU-University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190 Vienna, Austria., Laurent C; Biocatalysis and Biosensing Laboratory, Department of Food Science and Technology, BOKU-University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190 Vienna, Austria., Feroz S; Institute of Applied Synthetic Chemistry, TU Wien, Getreidemarkt 9, 1060 Vienna, Austria.; Department of Biosciences, College of Science, University of Hafr Al Batin, PO Box 1803, Hafr Al Batin, 39524, Saudi Arabia., P Goncalves LC; Institut de Chimie de Nice CRNS UMR7272, Université Côte d'Azur, 28 Avenue Valrose, 06108 Nice, France., Rial DV; Área Biología Molecular, Departamento de Ciencias Biológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario (UNR), and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Suipacha 531, S2002LRK Rosario, Argentina., Mihovilovic MD; Institute of Applied Synthetic Chemistry, TU Wien, Getreidemarkt 9, 1060 Vienna, Austria., Bommarius AS; School of Chemical & Biomolecular Engineering, Engineered Biosystems Building (EBB), Georgia Institute of Technology, 950 Atlantic Drive, N.W., Atlanta, Georgia 30332, United States., Ludwig R; Biocatalysis and Biosensing Laboratory, Department of Food Science and Technology, BOKU-University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190 Vienna, Austria., Oostenbrink C; Institute of Molecular Modeling and Simulation, University of Natural Resources and Life Sciences, 1190 Vienna, Austria., Rudroff F; Institute of Applied Synthetic Chemistry, TU Wien, Getreidemarkt 9, 1060 Vienna, Austria.
المصدر: ACS catalysis [ACS Catal] 2022 Oct 07; Vol. 12 (19), pp. 11761-11766. Date of Electronic Publication: 2022 Sep 13.
نوع المنشور: Journal Article
اللغة: English
بيانات الدورية: Publisher: American Chemical Society Country of Publication: United States NLM ID: 101562209 Publication Model: Print-Electronic Cited Medium: Print ISSN: 2155-5435 (Print) NLM ISO Abbreviation: ACS Catal Subsets: PubMed not MEDLINE
أسماء مطبوعة: Original Publication: Washington, DC : American Chemical Society, c2011-
مستخلص: The typically low thermodynamic and kinetic stability of enzymes is a bottleneck for their application in industrial synthesis. Baeyer-Villiger monooxygenases, which oxidize ketones to lactones using aerial oxygen, among other activities, suffer particularly from these instabilities. Previous efforts in protein engineering have increased thermodynamic stability but at the price of decreased activity. Here, we solved this trade-off by introducing mutations in a cyclohexanone monooxygenase from Acinetobacter sp., guided by a combination of rational and structure-guided consensus approaches. We developed variants with improved activity (1.5- to 2.5-fold) and increased thermodynamic (+5 °C T m ) and kinetic stability (8-fold). Our analysis revealed a crucial position in the cofactor binding domain, responsible for an 11-fold increase in affinity to the flavin cofactor, and explained using MD simulations. This gain in affinity was compatible with other mutations. While our study focused on a particular model enzyme, previous studies indicate that these findings are plausibly applicable to other BVMOs, and possibly to other flavin-dependent monooxygenases. These new design principles can inform the development of industrially robust, flavin-dependent biocatalysts for various oxidations.
Competing Interests: The authors declare no competing financial interest.
(© 2022 The Authors. Published by American Chemical Society.)
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تواريخ الأحداث: Date Created: 20221017 Latest Revision: 20221019
رمز التحديث: 20240628
مُعرف محوري في PubMed: PMC9552169
DOI: 10.1021/acscatal.2c03225
PMID: 36249873
قاعدة البيانات: MEDLINE
الوصف
تدمد:2155-5435
DOI:10.1021/acscatal.2c03225