ABSTRACT
The sulfhydryl oxidase Ero1 oxidizes protein disulfide isomerase (PDI), which in turn catalyzes disulfide formation in proteins folding in the endoplasmic reticulum (ER). The extent to which other members of the PDI family are oxidized by Ero1 and thus contribute to net disulfide formation in the ER has been an open question. The yeast ER contains four PDI family proteins with at least one potential redox-active cysteine pair. We monitored the direct oxidation of each redox-active site in these proteins by yeast Ero1p in vitro. In this study, we found that the Pdi1p amino-terminal domain was oxidized most rapidly compared with the other oxidoreductase active sites tested, including the Pdi1p carboxyl-terminal domain. This observation is consistent with experiments conducted in yeast cells. In particular, the amino-terminal domain of Pdi1p preferentially formed mixed disulfides with Ero1p in vivo, and we observed synthetic lethality between a temperature-sensitive Ero1p variant and mutant Pdi1p lacking the amino-terminal active-site disulfide. Thus, the amino-terminal domain of yeast Pdi1p is on a preferred pathway for oxidizing the ER thiol pool. Overall, our results provide a rank order for the tendency of yeast ER oxidoreductases to acquire disulfides from Ero1p.
Subject(s)
Endoplasmic Reticulum/enzymology , Glycoproteins/metabolism , Oxidoreductases Acting on Sulfur Group Donors/metabolism , Oxygen/metabolism , Protein Disulfide-Isomerases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Catalysis , Catalytic Domain , Disulfides/chemistry , Flavins/chemistry , Genetic Variation , Glutathione/metabolism , Mutation , Oxygen/chemistry , Protein Folding , Protein Structure, Tertiary , Saccharomyces cerevisiae/genetics , TemperatureABSTRACT
The ERV/ALR sulfhydryl oxidase domain is a versatile module adapted for catalysis of disulfide bond formation in various organelles and biological settings. Its four-helix bundle structure juxtaposes a Cys-X-X-Cys dithiol/disulfide motif with a bound flavin adenine dinucleotide (FAD) cofactor, enabling transfer of electrons from thiol substrates to non-thiol electron acceptors. ERV/ALR family members contain an additional di-cysteine motif outside the four-helix-bundle core. Although the location and context of this "shuttle" disulfide differs among family members, it is proposed to perform the same basic function of mediating electron transfer from substrate to the enzyme active site. We have determined by X-ray crystallography the structure of AtErv1, an ERV/ALR enzyme that contains a Cys-X4-Cys shuttle disulfide and oxidizes thioredoxin in vitro, and compared it to ScErv2, which has a Cys-X-Cys shuttle and does not oxidize thioredoxin at an appreciable rate. The AtErv1 shuttle disulfide is in a region of the structure that is disordered and thus apparently mobile and exposed. This feature may facilitate access of protein substrates to the shuttle disulfide. To test whether the shuttle disulfide region is modular and can confer on other enzymes oxidase activity toward new substrates, we generated chimeric enzyme variants combining shuttle disulfide and core elements from AtErv1 and ScErv2 and monitored oxidation of thioredoxin by the chimeras. We found that the AtErv1 shuttle disulfide region could indeed confer thioredoxin oxidase activity on the ScErv2 core. Remarkably, various chimeras containing the ScErv2 Cys-X-Cys shuttle disulfide were found to function efficiently as well. Since neither the ScErv2 core nor the Cys-X-Cys motif is therefore incapable of participating in oxidation of thioredoxin, we conclude that wild-type ScErv2 has evolved to repress activity on substrates of this type, perhaps in favor of a different, as yet unknown, substrate.
Subject(s)
Disulfides , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Plant Proteins/chemistry , Plant Proteins/metabolism , Protein Structure, Tertiary , Arabidopsis/enzymology , Crystallography, X-Ray , Directed Molecular Evolution , Models, Molecular , Molecular Sequence Data , Oxidation-Reduction , Oxidoreductases/genetics , Plant Proteins/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence AlignmentABSTRACT
Ero1p is a key enzyme in the disulfide bond formation pathway in eukaryotic cells in both aerobic and anaerobic environments. It was previously demonstrated that Ero1p can transfer electrons from thiol substrates to molecular oxygen. However, the fate of electrons under anaerobic conditions and the final fate of electrons under aerobic conditions remained obscure. To address these fundamental issues in the Ero1p mechanism, we studied the transfer of electrons from recombinant yeast Ero1p to various electron acceptors. Under aerobic conditions, reduction of molecular oxygen by Ero1p yielded stoichiometric hydrogen peroxide. Remarkably, we found that reduced Ero1p can transfer electrons to a variety of small and macromolecular electron acceptors in addition to molecular oxygen. In particular, Ero1p can catalyze reduction of exogenous FAD in solution. Free FAD is not required for the catalysis of dithiol oxidation by Ero1p, but it is sufficient to drive disulfide bond formation under anaerobic conditions. These findings provide insight into mechanisms for regenerating oxidized Ero1p and maintaining disulfide bond formation under anaerobic conditions in the endoplasmic reticulum.
