biostudies-literatureBarone MEuropean Cooperation in Science and TechnologyMinistero dell'Università e della Ricerca107810https://www.ebi.ac.uk/biostudies/studies/S-EPMC11530802biostudies-literatureUnknown300(11)Prenylcysteine oxidases (PCYOXs) metabolize prenylated cysteines produced by protein degradation. They utilize oxygen as a co-substrate to produce free cysteine, an aldehyde, and hydrogen peroxide through the unusual oxidation of a thioether bond. In this study, we explore the evolution, structure, and mechanism of the two mammalian PCYOXs. A gene duplication event in jawed vertebrates originated in these two paralogs. Both enzymes are active on farnesyl- and geranylgeranylcysteine, but inactive on molecules with shorter prenyl groups. Kinetics experiments outline a mechanism where flavin reduction and re-oxidation occur rapidly without any detectable intermediates, with the overall reaction rate limited by product release. The experimentally determined three-dimensional structure of PCYOXThe Journal of biological chemistryEvolution, structure, and drug-metabolizing activity of mammalian prenylcysteine oxidases.PMC11530802P2022FESRRBarone MPizzorni LMattevi AMascotti MLFraaije MWfalseEvolution, structure, and drug-metabolizing activity of mammalian prenylcysteine oxidases.Prenylcysteine oxidases (PCYOXs) metabolize prenylated cysteines produced by protein degradation. They utilize oxygen as a co-substrate to produce free cysteine, an aldehyde, and hydrogen peroxide through the unusual oxidation of a thioether bond. In this study, we explore the evolution, structure, and mechanism of the two mammalian PCYOXs. A gene duplication event in jawed vertebrates originated in these two paralogs. Both enzymes are active on farnesyl- and geranylgeranylcysteine, but inactive on molecules with shorter prenyl groups. Kinetics experiments outline a mechanism where flavin reduction and re-oxidation occur rapidly without any detectable intermediates, with the overall reaction rate limited by product release. The experimentally determined three-dimensional structure of PCYOX2024-01-01T00:00:00Z2024 Nov2026-06-02T22:13:55.907Z2025-04-06T14:24:52.729ZS-EPMC115308023932201610.1016/j.jbc.2024.107810