Exploring the Biosynthetic Landscape and Reaction Mechanisms of Non-Canonical Cytochrome P450 Enzymes

Researchers from Princeton University are investigating a newly discovered family of enzymes to determine how subtle changes in protein structure give rise to fundamentally new chemical reactions and to enable the development of next-generation biocatalysts. Enzymes are nature's catalysts, performing chemical transformations that sustain life and provide valuable tools for biotechnology, medicine, and sustainable chemical synthesis. The current project focuses on cytochrome P450 (CYP) enzymes, a large family of biocatalysts that carry out an extraordinary range of oxidative reactions involved in antibiotic production, natural product biosynthesis, and environmental chemistry. All characterized CYPs rely on a conserved cysteine residue to coordinate a heme-iron and activate oxygen, thereby forming a key reactive intermediate. However, genome mining has recently uncovered nearly 1,000 previously unrecognized non-canonical CYPs (ncCYPs) that instead use chemically distinct amino acid ligands, suggesting that nature has evolved fundamentally different strategies for oxidative chemistry. The research team will combine computational and experimental approaches to determine how these enzymes function, identify the metabolic pathways they support, and define the novel chemical reactions they catalyze. Their discoveries will provide insights into heme enzyme chemistry, reveal previously unknown mechanisms of microbial metabolism, and establish new enzyme platforms for biotechnology. The project will also broaden participation in STEM by providing interdisciplinary research experiences and sustained mentorship for undergraduate students, graduate students, postdoctoral researchers, and faculty from under-resourced institutions while strengthening partnerships that expand access to modern biochemical research. Building on the recent discovery of ~1,000 ncCYPs, the project will investigate representative enzymes spanning the newly identified diversity of amino acid ligands. Recombinant proteins will be expressed and purified for spectroscopic, biophysical, and enzymatic analyses to determine how alternative ligands alter the electronic structure of the heme cofactor, oxygen activation, and catalytic reactivity. Biosynthetic gene clusters encoding representative ncCYPs will be identified and experimentally characterized through heterologous pathway reconstruction and native host studies to establish the physiological roles of these enzymes in metabolism. Structural studies, complemented by structure-guided mutagenesis, will define the molecular determinants of substrate recognition and catalysis. Finally, representative ncCYPs will be screened against biological and synthetic substrate libraries to discover new oxidative transformations and evaluate their utility as versatile biocatalysts. Together, these integrated studies will establish the fundamental principles governing ncCYPs, illuminate new pathways for enzyme evolution and microbial natural product biosynthesis, and provide a foundation for developing oxidative biocatalysts with broad applications in biotechnology. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.