Cytochrome P450 Surface Domains Prevent the β-Carotene Monohydroxylase CYP97H1 of Euglena gracilis from Acting as a Dihydroxylase

Abstract

Molecular biodiversity results from branched metabolic pathways driven by enzymatic regioselectivities. An additional complexity occurs in metabolites with an internal structural symmetry, offering identical extremities to the enzymes. For example, in the terpene family, β-carotene presents two identical terminal closed-ring structures. Theses cycles can be hydroxylated by cytochrome P450s from the CYP97 family. Two sequential hydroxylations lead first to the formation of monohydroxylated β-cryptoxanthin and subsequently to that of dihydroxylated zeaxanthin. Among the CYP97 dihydroxylases, CYP97H1 from Euglena gracilis has been described as the only monohydroxylase. This study aims to determine which enzymatic domains are involved in this regioselectivity, conferring unique monohydroxylase activity on a substrate offering two identical sites for hydroxylation. We explored the effect of truncations, substitutions and domain swapping with other CYP97 members and found that CYP97H1 harbours a unique N-terminal globular domain. This CYP97H1 N-terminal domain harbours a hydrophobic patch at the entrance of the substrate channel, which is involved in the monohydroxylase activity of CYP97H1. This domain, at the surface of the enzyme, highlights the role of distal and non-catalytic domains in regulating enzyme specificity.

Keywords: asymmetric catalysis; carotenoids; cytochrome P450; protein engineering; regioselectivity; β-cryptoxanthin.

Figure 1

Hydroxylations in the β-carotene and α-carotene pathways with characterised mono- and dihydroxylases. β-cyclase and ε-cyclase refer to lycopene cyclase, leading to β or ε cyclisation, respectively. BCH1 refers to Bacterial Carotene Hydroxylase 1 from A. thaliana. CYP97A refers to the cytochrome P450 97 family, clan A. CYP97H1 originates from E. gracilis; CYP97A3 and CYP97C1 originate from A. thaliana.

Figure 2

(A): CYP97 alignment and key P450s domains of CYP97H1 from E. gracilis, CYP97A3, CYP97B3, CYP97C1 from A. thaliana, CYP97A4, CYP97B4, CYP97C2 from O. sativa, _CtCYP97 from C. unshiu and CYP97 K15747 from C. maxima. (B): Tridimensional alignment of CYP97A3 from A. thaliana (grey, heme in red) and CYP97H1 from E. gracilis (orange). The three specific regions of CYP97H1 are highlighted: F′-G′ loop (green), substrate channel hydrophobicity (yellow) and N-terminal extension (blue).

Figure 3

(A): CYP97 family alignment with a focus on the F′-G′ region; species and sequence references were extracted from the Uniprot database. (B): Alphafold2 modelling of the chimera _tCYP97H1_fullAt (CYP97H1 from E. gracilis is in gold, F′-G′ loop from A. thaliana is in green, and the heme from CYP97A3 alignment is in red). (C): In vivo carotenoid production in the strains expressing F′-G′ loop P450 chimeras.

Figure 4

(A): Full-length modelling of CYP97H1, with poor modelling of the N-terminal domain. The model was rotated horizontally at 90 °C compared to the CYP97H1 model of Figure 2B. The heme from the CYP97A3 crystal is represented in red. The N-terminal colouring represents the sequential truncated mutants assayed. (B): In vivo carotenoid production in strains expressing CYP97H1 N-terminal truncations.

Figure 5

(A): Docking of zeaxanthin (green) into CYP97H1. The specific CYP97H1 N-terminal domain (blue) harbours a substrate channel extension surrounded by hydrophobic residues (dark green), where the second hydroxyl group of zeaxanthin colocalizes. The CYP97H1 globular P450 domain (grey) harbours the catalytic heme from CYP97A3 (red) (B): In vivo carotenoid production (logarithmic scale) in strains expressing the CYP97H1 chimera with CYP97A3 residues or domains (pink) or with mutations in the hydrophobic patch (purple). Inset: ratio of zeaxanthin and β-cryptoxanthin in CYP97H1 and the F105A mutant, averaged over three experiments, with each including four biological replicates.