Rational Design of a Minimal and Highly Enriched CYP102A1 Mutant Library with Improved Regio‐, Stereo‐ and Chemoselectivity

ChemBioChem - Tập 10 Số 5 - Trang 853-861 - 2009
Alexander Seifert1, Sandra Vomund1, Katrin Grohmann2, Sebastian Kriening2, Vlada B. Urlacher1, Sabine Laschat2, Jürgen Pleiss1
1Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany,
2Institute of Organic Chemistry, University of Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany

Tóm tắt

AbstractMonooxygenase mutants: A minimal and highly enriched CYP102A1 mutant library was constructed by combining five hydrophobic amino acids in two positions. The library was screened with four different terpene substrates. Eleven variants demonstrated either a strong shift or improved regio‐ or stereoselectivity during oxidation of at least one substrate as compared to CYP102A1 wild type.magnified imageA minimal CYP102A1 mutant library of only 24 variants plus wild type was constructed by combining five hydrophobic amino acids (alanine, valine, phenylalanine, leucine and isoleucine) in two positions. Both positions are located close to the centre of the haem group. The first, position 87, has been shown to mediate substrate specificity and regioselectivity in CYP102A1. The second hotspot, position 328, was predicted to interact with all substrates during oxidation and has previously been identified by systematic analysis of 31 crystal structures and 6300 sequences of cytochrome P450 monooxygenases. By systematically altering the size of the side chains, a broad range of binding site shapes was generated. All variants were functionally expressed in E. coli. The library was screened with four terpene substrates geranylacetone, nerylacetone, (4R)‐limonene and (+)‐valencene. Only three variants showed no activity towards all four terpenes, while eleven variants demonstrated either a strong shift or improved regio‐ or stereoselectivity during oxidation of at least one substrate as compared to CYP102A1 wild type.

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ChemBioChem

Tập 10 Số 5

853-861

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