Structural studies of bacterial steroid-hydroxylating cytochrome P450 monooxygenases: Insights into the molecular basis of substrate specificity, selectivity and product formation

Ilona Jóźwik

Structural studies of bacterial steroid-hydroxylating cytochrome P450 monooxygenases: Insights into the molecular basis of substrate specificity, selectivity and product formation

Vertaalde titel van de bijdrage: Kristallografische studies van de 3D-structuren van bacteriële steroïd-hydroxylerende cytochroom P450 enzymen

Ilona Jóźwik

X-ray Structuurbepaling

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Cytochroom P450 monooxygenasen (P450s) zijn veelzijdige enzymen die gebruikt worden voor de specifieke oxidatie van chemische verbindingen, in een één-staps, milieuvriendelijke reactie. Echter, voor succesvolle toepassing van deze enzymen als biokatalysator in de fijnchemische en farmaceutische industrie is het dikwijls nodig om de activiteit van deze enzymen te verhogen, of hun regio- en/of stereoselectiviteit aan te passen. Bacteriële P450's zijn in het algemeen gemakkelijker te verbeteren dan P450s uit hogere organismen, vooral vanwege hun goede oplosbaarheid en betere selectiviteit. Eiwitkristallografie is een nuttige techniek om te bepalen welke aminozuren belangrijk zijn voor de functie en eigenschappen van een enzym. Deze techniek werd gebruikt om de actieve centra van drie P450 enzymen in detail te onderzoeken.

Dit proefschrift beschrijft de structuurbepaling en analyse van de driedimensionale structuur van drie biotechnologisch belangrijke bacteriële P450-enzymen (en/of mutante vormen), die steroïden en steroïde-gerelateerde verbindingen, zoals testosteron en vitamine D3, hydroxyleren. Twee Bacillus megaterium enzymen zijn onderzocht (CYP109E1, CYP109A2), alsmede twee varianten van de Sorangium cellulosum So ce56 CYP260A1. De opgehelderde structuren, in combinatie met biochemische experimenten en computerberekeningen, gaven inzicht in de aminozuren die van belang zijn voor de regio- en stereoselectiviteit van de onderzochte enzymen. Deze inzichten zijn van essentieel belang voor het begrijpen en verbeteren van de functionele eigenschappen van de enzymen, en om hen aan te passen en geschikt te maken voor nieuwe toepassingen.

Vertaalde titel van de bijdrage	Kristallografische studies van de 3D-structuren van bacteriële steroïd-hydroxylerende cytochroom P450 enzymen
Originele taal-2	English
Kwalificatie	Doctor of Philosophy
Toekennende instantie	Rijksuniversiteit Groningen
Begeleider(s)/adviseur	Dijkstra, Bauke, Supervisor Thunnissen, Andy, Co-supervisor
Datum van toekenning	23-jun.-2017
Plaats van publicatie	[Groningen]
Uitgever	University of Groningen
Gedrukte ISBN's	978-90-367-9791-7
Elektronische ISBN's	978-90-367-9790-0
Status	Published - 2017

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Title and contentsFinal publisher's version, 2,12 MB
Chapter 1Final publisher's version, 29,3 MB
Chapter 2Final publisher's version, 10,5 MB
Chapter 3Final publisher's version, 3 MB
Chapter 4Final publisher's version, 12,2 MB
Chapter 5Final publisher's version, 9,85 MB
Chapter 6Final publisher's version, 650 KB
Brief thesis summariesFinal publisher's version, 174 KB
Acknowledgements PodziękowaniaFinal publisher's version, 239 KB
Complete thesisFinal publisher's version, 67,1 MB
PropositionsFinal publisher's version, 90,4 KB

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title = "Structural studies of bacterial steroid-hydroxylating cytochrome P450 monooxygenases: Insights into the molecular basis of substrate specificity, selectivity and product formation",

abstract = "Cytochrome P450 monooxygenases (P450s) are highly versatile enzymes that very specifically oxidize their substrates in a single environmentally friendly reaction step. However, to allow their successful application as biocatalysts in the fine chemical or pharmaceutical industry, it is often necessary to increase their enzymatic activity or adapt their regio- and/or stereo-selectivity. Bacterial P450s are in general easier to improve than their counterparts from higher organisms, due to their higher solubility and selectivity. Protein X-ray crystallography is a useful technique to identify the structural features that underlie protein function, and was applied here to deeply investigate the active sites of three bacterial P450 enzymes. This thesis presents the determination and analysis of 3D crystal structures of three P450s (and mutant forms) that are able to hydroxylate steroids and steroid-derived compounds like testosterone or vitamin D3. Two Bacillus megaterium enzymes were investigated (CYP109E1, CYP109A2), as well as two variants of the Sorangium cellulosum So ce56 CYP260A1. The overall conclusion emerging from this research is that the size and shape of the active site, together with the distribution of active site polar/apolar regions, constitute the molecular determinants in the P450 protein dictating which steroid substrates may bind and at what position they are hydroxylated (the observed regio- and stereo-selectivity). Important roles in substrate binding of several active site residues could be determined thanks to the elucidated structures and data obtained from computational investigations and biochemical experiments. The insights will help to improve the functional aspects of these proteins via future protein engineering.",

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T2 - Insights into the molecular basis of substrate specificity, selectivity and product formation

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N2 - Cytochrome P450 monooxygenases (P450s) are highly versatile enzymes that very specifically oxidize their substrates in a single environmentally friendly reaction step. However, to allow their successful application as biocatalysts in the fine chemical or pharmaceutical industry, it is often necessary to increase their enzymatic activity or adapt their regio- and/or stereo-selectivity. Bacterial P450s are in general easier to improve than their counterparts from higher organisms, due to their higher solubility and selectivity. Protein X-ray crystallography is a useful technique to identify the structural features that underlie protein function, and was applied here to deeply investigate the active sites of three bacterial P450 enzymes. This thesis presents the determination and analysis of 3D crystal structures of three P450s (and mutant forms) that are able to hydroxylate steroids and steroid-derived compounds like testosterone or vitamin D3. Two Bacillus megaterium enzymes were investigated (CYP109E1, CYP109A2), as well as two variants of the Sorangium cellulosum So ce56 CYP260A1. The overall conclusion emerging from this research is that the size and shape of the active site, together with the distribution of active site polar/apolar regions, constitute the molecular determinants in the P450 protein dictating which steroid substrates may bind and at what position they are hydroxylated (the observed regio- and stereo-selectivity). Important roles in substrate binding of several active site residues could be determined thanks to the elucidated structures and data obtained from computational investigations and biochemical experiments. The insights will help to improve the functional aspects of these proteins via future protein engineering.

AB - Cytochrome P450 monooxygenases (P450s) are highly versatile enzymes that very specifically oxidize their substrates in a single environmentally friendly reaction step. However, to allow their successful application as biocatalysts in the fine chemical or pharmaceutical industry, it is often necessary to increase their enzymatic activity or adapt their regio- and/or stereo-selectivity. Bacterial P450s are in general easier to improve than their counterparts from higher organisms, due to their higher solubility and selectivity. Protein X-ray crystallography is a useful technique to identify the structural features that underlie protein function, and was applied here to deeply investigate the active sites of three bacterial P450 enzymes. This thesis presents the determination and analysis of 3D crystal structures of three P450s (and mutant forms) that are able to hydroxylate steroids and steroid-derived compounds like testosterone or vitamin D3. Two Bacillus megaterium enzymes were investigated (CYP109E1, CYP109A2), as well as two variants of the Sorangium cellulosum So ce56 CYP260A1. The overall conclusion emerging from this research is that the size and shape of the active site, together with the distribution of active site polar/apolar regions, constitute the molecular determinants in the P450 protein dictating which steroid substrates may bind and at what position they are hydroxylated (the observed regio- and stereo-selectivity). Important roles in substrate binding of several active site residues could be determined thanks to the elucidated structures and data obtained from computational investigations and biochemical experiments. The insights will help to improve the functional aspects of these proteins via future protein engineering.

M3 - Thesis fully internal (DIV)

SN - 978-90-367-9791-7

PB - University of Groningen

CY - [Groningen]

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