Applied Microbiology and Biotechnology

, Volume 97, Issue 4, pp 1625–1635 | Cite as

Cytochrome P450 reductase from Candida apicola: versatile redox partner for bacterial P450s

  • Marco Girhard
  • Florian Tieves
  • Evelyne Weber
  • Martha Sophia Smit
  • Vlada B. Urlacher
Biotechnologically relevant enzymes and proteins
First Online:
Received:
Revised:
Accepted:

Abstract

Candida apicola belongs to a group of yeasts producing surface-active glycolipids consisting of sophorose and long-chain (ω)- or (ω-1)-hydroxy fatty acids. Hydroxylation of the fatty acids in this strain is likely catalyzed by cytochrome P450 monooxygenases (P450), which require reducing equivalents delivered via a cytochrome P450-diflavin reductase (CPR). We herein report cloning and characterization of the cpr gene from C. apicola ATCC 96134. The gene encoding a protein of 687 amino acids was cloned in Escherichia coli and the enzyme was expressed in functional form after truncation of its N-terminal putative membrane anchor. The truncated recombinant protein showed cytochrome c reducing activity (K M of 13.8 μM and k cat of 1,915 per minute). Furthermore, we herein demonstrate to our best knowledge for the first time the use of a eukaryotic CPR to transfer electrons to bacterial P450s (namely CYP109B1 and CYP154E1). Cloning and characterization of this CPR therefore is not only an important step in the study of the P450 systems of C. apicola, but also provides a versatile redox partner for the characterization of other bacterial P450s with appealing biotechnological potential. The GenBank accession number of the sequence described in this article is JQ015264.

Keywords

Candida apicola Cytochrome P450-diflavin reductase CPR P450 Heterologous expression 
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Notes

Acknowledgments

The authors wish to thank Dr. Akira Arisawa (Mercian Corporations, Iwata, Japan) for kind provision of a plasmid containing the gene coding for CYP154E1, and Prof. Rita Bernhardt (Saarland University) for kind providing of AdR and Adx. The help of Anna Romankiewicz (Universtät Stuttgart, Germany) and Clemens von Bühler (Heinrich-Heine University Düsseldorf, Germany) for cloning and purification of CYP154E1 is acknowledged. V.B.U. and E.W. wish to acknowledge partial funding of this work by Deutsche Forschungsgemeinschaft (SFB706) and Ministerium für Wissenschaft, Forschung und Kunst Baden-Württemberg.

Supplementary material

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ESM 1 (PDF 330 kb)

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Marco Girhard
    • 1
  • Florian Tieves
    • 1
  • Evelyne Weber
    • 2
  • Martha Sophia Smit
    • 3
  • Vlada B. Urlacher
    • 1
  1. 1.Institute of BiochemistryHeinrich-Heine University DüsseldorfDüsseldorfGermany
  2. 2.Institute of Technical BiochemistryUniversität StuttgartStuttgartGermany
  3. 3.Department of Microbial, Biochemical and Food BiotechnologyUniversity of the Free StateBloemfonteinSouth Africa

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