Evolution, structure, and drug-metabolizing activity of mammalian prenylcysteine oxidases

Abstract

Prenylcysteine oxidases (PCYOXs) metabolize prenylatedcysteines produced by protein degradation. They utilize oxygenas a co-substrate to produce free cysteine, an aldehyde, andhydrogen peroxide through the unusual oxidation of a thio-ether bond. In this study, we explore the evolution, structure,and mechanism of the two mammalian PCYOXs. A geneduplication event in jawed vertebrates originated in these twoparalogs. Both enzymes are active on farnesyl- and ger-anylgeranylcysteine, but inactive on molecules with shorterprenyl groups. Kinetics experiments outline a mechanismwhere flavin reduction and re-oxidation occur rapidly withoutany detectable intermediates, with the overall reaction ratelimited by product release. The experimentally determinedthree-dimensional structure of PCYOX1 reveals long and widetunnels leading from the surface to the flavin. They allow theisoprene substrate to curl up within the protein and position itsreactive cysteine group close to the flavin. A hydrophobic patchon the surface mediates membrane association, enabling directsubstrate and product exchange with the lipid bilayer.Leveraging established knowledge of flavoenzyme inhibition,we designed sub-micromolar PCYOX inhibitors. Additionally,we discovered that PCYOXs bind and slowly degrade salisirab,an anti-RAS compound. This activity suggests potential andpreviously unknown roles of PCYOXs in drug metabolism.