CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering**

ChemBioChem, vol. 22, pp. 1099–1110

Abstract

CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure-function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation.

Authors 9

1. Hein J. Wijma

   University of Groningen

   Affiliation as printed

   Department of Biochemistry Groningen Biomolecular Sciences and Biotechnology Institute University of Groningen Nijenborgh 4 9747 AG Groningen Netherlands

   Department of Biochemistry Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, Netherlands

2. Bastian Nicolai Aachen Biocatalysis Institute of Biotechnology

   RWTH Aachen University

   Affiliation as printed

   Biocatalysis, Institute of Biotechnology RWTH Aachen University Worringerweg 3 52074 Aachen Germany

   Biocatalysis, Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany

3. Jhon Alexander Rodriguez Buitrago

   Technische Universität Braunschweig

   Affiliation as printed

   Institute for Biochemistry, Biotechnology and Bioinformatics Technische Universität Braunschweig Spielmannstr. 7 38106 Braunschweig Germany

4. Thomas Klünemann

   Helmholtz Centre for Infection Research

   Affiliation as printed

   Structure and Function of Proteins Helmholtz Centre for Infection Research Inhoffenstr. 7 38124 Braunschweig Germany

   Structure and Function of Proteins, Helmholtz Centre for Infection Research, Inhoffenstr. 7, 38124 Braunschweig, Germany

5. Agustina Vila Aachen Biocatalysis Institute of Biotechnology

   RWTH Aachen University

   Affiliation as printed

   Biocatalysis, Institute of Biotechnology RWTH Aachen University Worringerweg 3 52074 Aachen Germany

   Biocatalysis, Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany

6. Patrick Schrepfer

   Technische Universität Braunschweig

   Affiliation as printed

   Institute for Biochemistry, Biotechnology and Bioinformatics Technische Universität Braunschweig Spielmannstr. 7 38106 Braunschweig Germany

7. Wulf Blankenfeldt

   Helmholtz Centre for Infection Research · Technische Universität Braunschweig

   Affiliation as printed

   Institute for Biochemistry, Biotechnology and Bioinformatics Technische Universität Braunschweig Spielmannstr. 7 38106 Braunschweig Germany

   Structure and Function of Proteins Helmholtz Centre for Infection Research Inhoffenstr. 7 38124 Braunschweig Germany

   Structure and Function of Proteins, Helmholtz Centre for Infection Research, Inhoffenstr. 7, 38124 Braunschweig, Germany

8. Dick B. Janssen

   University of Groningen

   Affiliation as printed

   Department of Biochemistry Groningen Biomolecular Sciences and Biotechnology Institute University of Groningen Nijenborgh 4 9747 AG Groningen Netherlands

   Department of Biochemistry Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, Netherlands

9. Anett Schallmey corresponding

   Technische Universität Braunschweig

   Affiliation as printed

   Institute for Biochemistry, Biotechnology and Bioinformatics Technische Universität Braunschweig Spielmannstr. 7 38106 Braunschweig Germany