Oxireductive Character And Potential Targets Of AtDsbA

Regulatory protein disulfide bonds serve as key signaling elements and theformation and the reduction reactions of the disulfides are specifically catalyzed invivo. In chloroplasts, regulatory disulfides are preferentially reduced by the dithiolreductant, thioredoxin, but their oxidant counterpart is yet to be identified. A novelfusion protein in Arabidopsis chloroplast encoded by gene At4g35760which namedAtVKOR-DsbA was found to be involved in disulfide formation. The gene productcontains two distinct domains: an integral membrane domain homologous to thecatalytic subunit of mammalian vitamin K epoxide reductase (VKOR) and a solubleDsbA-like domain.However，the function of foldase needed further investigation. Theproteins intreracted with AtVKOR-DsbA haven’t been identified yet.In this paper, the activities of reductase, oxygenase and isomerase of AtDsbAwas found from researches of insulin reduction assay, denatured RNase A refoldingassay and scrambled RNase A refolding assay.Four cystiens in AtDsbA was found esstial for the activity of AtVKOR-DsbA,which cysteins were the functional cysteins for AtDsbA hadn’t been investigated yet.The cysteins in CXXC motif were found to be the functional cysteins throughsite-mutant by quick-change method.The substrate of AtDsbA was captured by affinity chromatography with thehistidine-tagged moncystein (WCXXA) mutant AtDsbA, and were identified byprotein mass fingerprinting (PMF) and database searching. Two potential targetproteins were identified from the protein complexes captured. One was encoded byAt2g27620, a homologue of ubiquitin carboxy terminal hydrolase (UCH), which is akind of deubiquitinating enzymes. Yet the other was encoded by At4g31390, ahomologue of a protein kinase, related to the quoin synthesis.