Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 284, Issue 1, Pages 199-206Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M806645200
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Funding
- Chinese Ministry of Science and Technology [2006CB910903, 2006CB806508, 2006CB910301]
- Chinese Natural Science Foundation [30620130109]
- UK BBSRC (Biotechnology and Biological Sciences Research Council) [BB/D017807/1, PA1339]
- BBSRC [BB/D017807/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/D017807/1] Funding Source: researchfish
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Protein-disulfide isomerase (PDI), a critical enzyme responsible for oxidative protein folding in the eukaryotic endoplasmic reticulum, is composed of four thioredoxin domains a, b, b', a', and a linker x between b' and a'. Ero1-L alpha, an oxidase for human PDI (hPDI), has been determined to have one molecular flavin adenine dinucleotide (FAD) as its prosthetic group. Oxygen consumption assays with purified recombinant Ero1-L alpha revealed that it utilizes oxygen as a terminal electron acceptor producing one disulfide bond and one molecule of hydrogen peroxide per dioxygen molecule consumed. Exogenous FAD is not required for recombinant Ero1-L alpha activity. By monitoring the reactivation of denatured and reduced RNase A, we reconstituted the Ero1-L alpha/hPDI oxidative folding system in vitro and determined the enzymatic activities of hPDI in this system. Mutagenesis studies suggested that the a' domain of hPDI is much more active than the a domain in Ero1-L alpha-mediated oxidative folding. A domain swapping study revealed that one catalytic thioredoxin domain to the C-terminal of bb' x, whether a or a' , is essential in Ero1-L alpha-mediated oxidative folding. These data, combined with a pull-down assay and isothermal titration calorimetry measurements, enabled the minimal element for binding with Ero1-L alpha be mapped to the b' xa' fragment of hPDI.
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