Functional characterization of a soluble NADPH-cytochrome P450 reductase from Fusarium graminearum

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  • Thomas Etzerodt
  • ,
  • Karl Wetterhorn, University of Wisconsin-Madison
  • ,
  • Giuseppe Dionisio
  • Ivan Rayment, University of Wisconsin-Madison

Fusarium head blight is a devastating disease in wheat caused by some fungal pathogens of the Fusarium genus mainly F. graminearum, due to accumulation of toxic trichothecenes. Most of the trichothecene biosynthetic pathway has been mapped, although some proteins of the pathway remain uncharacterized, including an NADPH-cytochrome P450 reductase. We subcloned a F. graminearum cytochrome P450 reductase that might be involved in the trichothecene biosynthesis. It was expressed heterologously in E. coli as N-terminal truncated form with an octahistidine tag for purification. The construct yielded a soluble apoprotein and its incubation with flavins yielded the corresponding monomeric holoprotein. It was characterized for activity in the pH range 5.5–9.5, using thiazolyl blue tetrazolium bromide (MTT) or cytochrome c as substrates. Binding of the small molecule MTT was weaker than for cytochrome c, however, the rate of MTT reduction was faster. Contrary to other studies of cytochrome reductase proteins, MTT reduction proceeded in a cooperative manner in our studies. Optimum kinetic activity was found at pH 7.5–8.5 for bothMTT and cytochrome c. This is the first paper presenting characterization of a cytochrome P450 reductase from F. graminearum which most likely is involved in mycotoxin biosynthesis or some primary metabolic pathway such as sterol biosynthesis in F. graminearum.

Original languageEnglish
JournalProtein Expression and Purification
Volume138
Pages (from-to)69-75
Number of pages7
ISSN1046-5928
DOIs
Publication statusPublished - 1 Oct 2017

    Research areas

  • Cytochrome c, Enzyme kinetics, Sterol biosynthesis, Thiazolyl blue tetrazolium bromide, Trichothecenes

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