Format

Send to

Choose Destination
Biochim Biophys Acta Proteins Proteom. 2018 Jan;1866(1):2-10. doi: 10.1016/j.bbapap.2017.07.008. Epub 2017 Jul 19.

Insights into the functional properties of the marneral oxidase CYP71A16 from Arabidopsis thaliana.

Author information

1
Institute of Biochemistry, Heinrich-Heine University Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany; Cluster of Excellence on Plant Sciences, Heinrich-Heine University Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany.
2
Institute of Biochemistry, Heinrich-Heine University Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany.
3
Institute of Biochemistry, Heinrich-Heine University Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany; Cluster of Excellence on Plant Sciences, Heinrich-Heine University Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany. Electronic address: vlada.urlacher@uni-duesseldorf.de.

Abstract

The Arabidopsis thaliana gene encoding CYP71A16 is part of the gene cluster for the biosynthesis and modification of the triterpenoid marneral. Previous investigations of A. thaliana have revealed that CYP71A16 catalyzes marneral oxidation, while it also can accept marnerol as substrate. The aim of the present study was to investigate functional properties of CYP71A16 in vitro. For this purpose, heterologous expression of a N-terminally modified version of CYP71A16 was established in Escherichia coli, which yielded up to 50mgL-1 recombinant enzyme. The enzyme was purified and activity was reconstituted in vitro with different redox partners. A heterologous bacterial redox partner system consisting of the flavodoxin YkuN from Bacillus subtilis and the flavodoxin reductase Fpr from E. coli clearly outperformed the cytochrome P450 reductase ATR2 from A. thaliana in supporting the CYP71A16-mediated hydroxylation of marnerol. Substrate binding experiments with CYP71A16 revealed a dissociation constant KD of 225μM for marnerol. CYP71A16 catalyzed the hydroxylation of marnerol to 23-hydroxymarnerol with a KM of 142μM and a kcat of 3.9min-1. Furthermore, GC/MS analysis revealed an as of yet unidentified overoxidation product of this in vitro reaction. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

KEYWORDS:

ATR2; CYP71A16; E. coli; Heterologous expression; Marnerol; Plant triterpenoid

PMID:
28734978
DOI:
10.1016/j.bbapap.2017.07.008
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center