Browsing by Author "Gricman, Lukasz"

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    Strategies for rational protein engineering of cytochrome P450 monooxygenase systems
    (2015) Gricman, Lukasz; Pleiss, Jürgen (Prof. Dr.)
    Cytochrome P450 monooxygenases (CYPs) constitute a large and diverse protein family. Because of their ability to introduce molecular oxygen into non-activated C-H bonds, CYPs are interesting enzymes for synthetic applications. However, stability, selectivity and activity are often obstacles prohibiting those enzymes from being used in industrial processes. Thus, novel protein engineering strategies for overcoming all those bottlenecks are required. In this work, a comprehensive guide to protein engineering of CYPs covering those three aspects was established to aid rational protein engineering of enzymes with and without known structure. The described protein engineering strategies are based on new insights into CYPs, which were gathered from incorporation of new functionalities into and analyses of the Cytochrome P450 Engineering Database (www.CYPED.BioCatNet.de), as well as molecular modeling of stereoselectivity in CYP101A1 from Pseudomonas putida. The described strategy for improving protein stability is based on the insights gathered from a systematic sequence-based protein stabilization of bovine adrenodoxin reductase. The presented strategies for protein engineering of selectivity are based on the identification of universal selectivity determining positions by systematic literature mining and on new insights from molecular modeling of stereoselectivity. The strategies for protein engineering of activity are based on the amino acid conservation analysis of CYPs, identification of the redox partner interaction sites, and a strategy for re-designing linker regions in artificial CYP fusion systems.