@article{mbs:/content/journal/micro/10.1099/mic.0.000569, author = "Hibender, Stijntje and Landeta, Cristina and Berkmen, Mehmet and Beckwith, Jon and Boyd, Dana", title = "Aeropyrum pernix membrane topology of protein VKOR promotes protein disulfide bond formation in two subcellular compartments", journal= "Microbiology", year = "2017", volume = "163", number = "12", pages = "1864-1879", doi = "https://doi.org/10.1099/mic.0.000569", url = "https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.000569", publisher = "Microbiology Society", issn = "1465-2080", type = "Journal Article", keywords = "Disulfide bond formation", keywords = "archaea", keywords = "membrane topology", keywords = "VKOR", abstract = "Disulfide bonds confer stability and activity to proteins. Bioinformatic approaches allow predictions of which organisms make protein disulfide bonds and in which subcellular compartments disulfide bond formation takes place. Such an analysis, along with biochemical and protein structural data, suggests that many of the extremophile Crenarachaea make protein disulfide bonds in both the cytoplasm and the cell envelope. We have sought to determine the oxidative folding pathways in the sequenced genomes of the Crenarchaea, by seeking homologues of the enzymes known to be involved in disulfide bond formation in bacteria. Some Crenarchaea have two homologues of the cytoplasmic membrane protein VKOR, a protein required in many bacteria for the oxidation of bacterial DsbAs. We show that the two VKORs of Aeropyrum pernix assume opposite orientations in the cytoplasmic membrane, when expressed in E. coli. One has its active cysteines oriented toward the E. coli periplasm (ApVKORo) and the other toward the cytoplasm (ApVKORi). Furthermore, the ApVKORo promotes disulfide bond formation in the E. coli cell envelope, while the ApVKORi promotes disulfide bond formation in the E. coli cytoplasm via a co-expressed archaeal protein ApPDO. Amongst the VKORs from different archaeal species, the pairs of VKORs in each species are much more closely related to each other than to the VKORs of the other species. The results suggest two independent occurrences of the evolution of the two topologically inverted VKORs in archaea. Our results suggest a mechanistic basis for the formation of disulfide bonds in the cytoplasm of Crenarchaea.", }