Analysis of substrate specificity of cytochrome P450 monooxygenases involved in trichothecene toxin biosynthesis

Authors: CARDOZA, ROSA - University Of Leon; McCormick, Susan; MARTINEZ-REYES, NATALIA - University Of Leon; RODRÍGUEZ-FERNÁNDEZ, JOAQUÍN - University Of Leon; Busman, Mark; Proctor, Robert; GUTIERREZ, SANTIAGO - University Of Leon

Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/6/2023
Publication Date: 1/6/2024
Citation: Cardoza, R.E., McCormick, S.P., Martinez-Reyes, N., Rodríguez-Fernández, J., Busman, M., Proctor, R.H., Gutiérrez, S. 2024. Analysis of substrate specificity of cytochrome P450 monooxygenases involved in trichothecene toxin biosynthesis. Applied Microbiology and Biotechnology. https://doi.org/10.1007/s00253-023-12950-1.
DOI: https://doi.org/10.1007/s00253-023-12950-1

Interpretive Summary: Trichothecenes are among the fungal toxins of most concern to food safety because of their frequent occurrence in cereal crops. Along with other fungal toxins, trichothecenes contribute to the estimated annual losses of $0.5 – 5 billion to the agricultural economies of the U.S. and Canada. Multiple strategies are being developed to combat trichothecene contamination in crops. Among the most promising is enzymatic detoxification. However, one barrier to this approach is narrow substrate specificity; that is, the inability of enzymes to detoxify trichothecene molecules that differ in chemical structure. To overcome the barrier, ARS researchers in Peoria, Illinois, and researchers at the Ponferrada Campus of the University of Leon, Spain, searched for factors that contribute to narrow substrate specificity of fungal enzymes that modify trichothecenes. Their results indicate that an oxygen atom at position 3 of trichothecene molecules is a major contributor to narrow substrate specificity of the enzymes examined. These results will aid development of detoxification enzymes that reduce trichothecene contamination in crops and thereby reduce economic losses caused by the toxins.

Technical Abstract: Trichothecenes are a structurally diverse family of toxic secondary metabolites produced by certain species of multiple fungal genera. All trichothecene analogs share a core 12,13-epoxytrichothec-9-ene (EPT) structure but differ in presence, absence and types of substituents attached to various positions of EPT. Formation of some of the structural diversity begins early in the biosynthetic pathway such that some species have few trichothecene biosynthetic intermediates in common. Cytochrome P450 monooxygenases (P450s) play critical roles in formation trichothecene structural diversity. Within some species, relaxed substrate specificities of P450s allow individual orthologs of the enzymes to modify multiple trichothecene biosynthetic intermediates. It is not clear, however, whether the relaxed specificity is such that a P450 ortholog can modify intermediates that are not produced by the species in which the ortholog originates. To address this knowledge gap, we used a transformation-mediated genetic complementation analysis to assess the ability of trichothecene biosynthetic P450s from three fungi, Fusarium sporotrichioides, Trichoderma arundinaceum, and Paramyrothecium roridum, to compensate for the absence of native P450s in mutant strains of T. arundinaceum and F. sporotrichioides. The results indicate that only P450s from species that produce the same early EPT derivatives can compensate for the absence of a native P450 in the mutants. These results raise the question, have fungi with divergent trichothecene pathways acquired different P450s to catalyze the same modification because the enzymes are restricted in their abilities to use structurally dissimilar biosynthetic intermediates as substrates?