Dataset for: Functional Characterization and Substrate Scope Expansion of Novel Cytochrome P450 Homologues Using a Fdx-FdR Fusion System

This dataset contains he raw data and processed experimental data supporting the above publication.

Cytochrome P450 monooxygenases of the CYP153A family are promising biocatalysts for selective C–H hydroxylation, yet their application is often limited by restricted substrate scope and challenges in electron-transfer compatibility. In this study, four CYP153A homologues from Salinisphaeraceae bacterium, Sphingomonas sp. Ant H11, Acidovorax sp. CHX100, and a Gammaproteobacteria bacterium were identified through sequence analysis using CYP153A6 enzymes from Mycobacterium sp. HXN-1500 as references and functionally characterized. A fusion protein comprising ferredoxin and ferredoxin reductase (Fdx-FdR) from Mycobacterium sp. was rationally designed to provide a modular electron-transfer system. The purified fusion exhibited reductase activity toward DCIP with both NADPH and NADH as electron donors (3.2 and 3.6 µmol s⁻¹ nmol⁻¹, respectively). Functional electron transfer was demonstrated by productive coupling of the fusion to all CYP homologues, as confirmed by substrate-scope analysis. Substrate screening revealed pronounced in regioselectivity and substrate preference among the homologues. Molecular dynamics simulations and thermal unfolding experiments identified AcCYP as the most stable homologue (Tm = 60.2 °C), whereas the Fdx-FdR fusion displayed limited thermal stability (Tm = 38.7 °C), defining the operational window of the system. Together, these results demonstrate that a single Fdx-FdR fusion can efficiently support diverse CYP153A homologues while enabling distinct substrate-selectivity profiles. This work establishes a modular and efficient platform for reconstituting and optimizing CYP153A enzymes, expanding their potential in biocatalysis and synthetic biology.