Characterisation of the Self-Sufficient Cytochrome P450 CYP116B234 From Rhodococcus globerulus and Its Suggested Native Role in 2-Hydroxyphenylacetic Acid Metabolism

Abstract

Cytochromes P450 (P450s) are exceptional biocatalysts that enable the selective oxidation of unactivated C–H bonds using molecular oxygen. Typically, auxiliary redox partner proteins deliver electrons from NAD(P)H to the P450, enabling oxygen activation. However, associating native redox partners with P450s can be challenging, particularly when they are genomically separated. Self-sufficient P450s, where the P450 heme domain is naturally fused to redox partners, represent a simpler, single-protein system. Here, we present CYP116B234, a novel self-sufficient P450 from Rhodococcus globerulus, comprising fused heme and phthalate-family oxygenase reductase (PFOR) domains. The gene encoding CYP116B234 was codon-optimised for heterologous expression in E. coli and subsequently purified to homogeneity. Spectroelectrochemical analysis and electron paramagnetic resonance spectroscopy were performed to determine the redox potentials of the heme and associated Fe–S and FMN cofactors of the PFOR domain. CYP116B234 binds and efficiently oxidises the substituted aromatic compound 2-hydroxyphenylacetic acid (2-HPA) to homogentisic acid. Quantitative proteomics revealed the expression of CYP116B234 in R. globerulus grown on 2-HPA, suggesting a role in initiating 2-HPA degradation. This study presents a new addition to the self-sufficient CYP116 family and provides evidence for their native function.