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

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, beta-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 beta-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.

Automated summary

Molecular biodiversity is a result of enzymatic regioselectivities in branched metabolic pathways. Metabolites with internal structural symmetry, such as beta-carotene, offer identical extremities to enzymes, presenting additional complexity. This study investigates the unique monohydroxylase activity of CYP97H1, the only monohydroxylase among CYP97 dihydroxylases, in a substrate with two identical sites for hydroxylation. It is found that the N-terminal globular domain of CYP97H1, which contains a hydrophobic patch at the entrance of the substrate channel, is responsible for this regioselectivity.