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

Abstract

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.

Figure 1

Reactions catalysed by chalcone 3-hydroxylase and flavonoid 3′-hydroxylase.

Figure 2

Phylogenetic tree of available Asteraceae CYP75B amino acid sequences. The enzymes exhibiting chalcone hydroxylase activity are coloured red and the F3′H from Tagetes erecta showing weak chalcone hydroxylase activity is coloured orange. Note: The sequence of F3′H from Petunia hybrida (Q9SBQ9) has been included for comparison with F3′H from non-Asteraceae species.

Figure 3

Structural features of DvCH3H. (a) Sequence of DvCH3H. Secondary structure features were assigned using DSSP (Dictionary of Secondary Structures of Proteins) using the homology model of DvCH3H. Grey-shaded boxes highlight II, proline-rich hinge^,; III, oxygen-binding motif; IV, EXXR triad^,; V, the P450-pattern^,. The blue framed box highlights the hydrophobic membrane anchor and the red framed boxes highlight the substrate recognition sites 1–6 (SRS1-6)^–. (b) Homology model of DvCH3H. The SRS regions is colored dark blue and the heme is coloured red. The putative orientation in and on the membrane of the endoplasmic reticulum has been roughly estimated according to the discussion in the literature.

Figure 4

Unit operations necessary for the purification of recombinant DvCH3H. Investigated factors and ranges thereof are shown in the right column.

Figure 5

(a) SDS-PAGE and western blot and (b) SEC-chromatogram of the purified recombinant DvCH3H. Solid line indicates absorbance at 280 nm and the dashed line the absorbance at 425 nm, where the heme of the Tris-(2-carboxyethyl)-phosphin reduced recombinant DvCH3H has an absorbance maximum. The original SDS-PAGE and western blot are shown in Supplementary Fig. S3.

Figure 6

Specific activity (v) of the recombinant membrane bound DvCH3H in dependence of the substrate concentration (S). The graphs show steady state kinetic measurements of DvCH3H using (a) (2S)-naringenin, (b) (2R)-naringenin, (c) racemic naringenin, (d) kaempferol and (e) apigenin as substrates.