First purified recombinant CYP75B including transmembrane helix with unexpected high substrate specificity to (2R)-naringenin

Sci Rep. 2022 May 20;12(1):8548. doi: 10.1038/s41598-022-11556-3.


Anthochlor pigments (chalcones and aurones) play an important role in yellow flower colourization, the formation of UV-honey guides and show numerous health benefits. The B-ring hydroxylation of chalcones is performed by membrane bound cytochrome P450 enzymes. It was assumed that usual flavonoid 3'-hydroxlases (F3'Hs) are responsible for the 3,4- dihydroxy pattern of chalcones, however, we previously showed that a specialized F3'H, namely chalcone 3-hydroxylase (CH3H), is necessary for the hydroxylation of chalcones. In this study, a sequence encoding membrane bound CH3H from Dahlia variabilis was recombinantly expressed in yeast and a purification procedure was developed. The optimized purification procedure led to an overall recovery of 30% recombinant DvCH3H with a purity of more than 84%. The enzyme was biochemically characterized with regard to its kinetic parameters on various substrates, including racemic naringenin, as well as its enantiomers (2S)-, and (2R)-naringenin, apigenin and kaempferol. We report for the first time the characterization of a purified Cytochrome P450 enzyme from the flavonoid biosynthesis pathway, including the transmembrane helix. Further, we show for the first time that recombinant DvCH3H displays a higher affinity for (2R)-naringenin than for (2S)-naringenin, although (2R)-flavanones are not naturally formed by chalcone isomerase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chalcone*
  • Chalcones* / metabolism
  • Cytochrome P-450 Enzyme System / metabolism
  • Flavanones* / chemistry
  • Flavonoids / metabolism
  • Plant Proteins / metabolism
  • Substrate Specificity


  • Chalcones
  • Flavanones
  • Flavonoids
  • Plant Proteins
  • Chalcone
  • Cytochrome P-450 Enzyme System
  • naringenin