Structural studies of bacterial steroid-hydroxylating cytochrome P450 monooxygenases Jóźwik, Ilona

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1 University of Groningen Structural studies of bacterial steroid-hydroxylating cytochrome P450 monooxygenases Jóźwik, Ilona IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2017 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Jóźwik, I. (2017). Structural studies of bacterial steroid-hydroxylating cytochrome P450 monooxygenases: Insights into the molecular basis of substrate specificity, selectivity and product formation [Groningen]: University of Groningen Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 Structural studies of bacterial steroid-hydroxylating cytochrome P450 monooxygenases Insights into the molecular basis of substrate specificity, selectivity and product formation Ilona K. Jóźwik 2017

3 Copyright Ilona K. Jóźwik 2017, Goningen, the Netherlands All rights reserved. No part of this thesis may be reproduced, stored in a retrieval system or transmitted in any form or by any means, without the permission of the author. Printed by: Ipskamp Drukkers B.V., Enschede Cover design by: Ilona K. Jóźwik Front cover: Crystal structure of substrate-free CYP109E1. Back cover: Crystallization drops that led to results described in this thesis. ISBN (printed version): ISBN (electronic version): The research described in this thesis was carried out in the Protein Crystallography group at the Groningen Biomolecular Sciences and Biotechnology Institute (GBB), Faculty of Science and Engineering, University of Groningen, the Netherlands. The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union s 7th Framework Programme (FP7/ ) under REA Grant Agreement (ITN P4FIFTY).

4 Structural studies of bacterial steroid-hydroxylating cytochrome P450 monooxygenases Insights into the molecular basis of substrate specificity, selectivity and product formation PhD thesis to obtain the degree of PhD at the University of Groningen on the authority of the Rector Magnificus Prof. E. Sterken and in accordance with the decision by the College of Deans. This thesis will be defended in public on Friday 23 June 2017 at hours by Ilona Katarzyna Jóźwik born on 6 May 1988 in Pabianice, Poland

5 Supervisor Prof. B.W. Dijkstra Co-supervisor Dr. A.M.W.H. Thunnissen Assessment Committee Prof. L. Dijkhuizen Prof. M.W. Fraaije Prof. G. Grogan

6 Steel is hardened in fire, not in water. Rodzicom i dziadkom

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8 Contents Chapter 1 General introduction Scope of this thesis 9 36 Chapter 2 Structural basis of steroid binding and oxidation by the cytochrome P450 CYP109E1 from Bacillus megaterium 49 Chapter 3 Chapter 4 Chapter 5 Characterization of cytochrome P450 CYP109E1 from Bacillus megaterium as a novel vitamin D 3 hydroxylase Crystal structure of the vitamin D 3 hydroxylating enzyme CYP109A2 Altering the regioselectivity of CYP260A1 for hydroxylation of progesterone by structure-based engineering Chapter 6 Summary, discussion and future perspectives 147 Brief thesis summaries 155 Acknowledgements / Podziękowania 159

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