Function And Essential Cysteins Analysis Of Arabidopsis VKOR

The disulfide bond is a covalent connection between two cysteines of a protein or two proteins. The disulfide bond formation is an important step of oxidized proteins to be mature, which can enhance the stability of proteins, avoid the proteins to be broken up, and reduce the degradation functions of protease. The disulfide bond is essential for the proteins to form correct structure and perform correct function. The disulfide bond formation system of E.coli has been studied clearly, and the electron transfer pathways of disulfide bond formation mainly achived througn DsbA and DsbB. In recent years, a new disulfide bond formation system has been discovered in microbiology(Vitamin K epoxide reductase system), and the Vitamin K epoxide reductase(VKOR) is the key component of this system. The homologous proteins of VKOR are exist in many higher plants, but whether the VKOR system is a general disulfide bond formation system is still unknown. We selected Arabidopsis as the target to study the disulfide bond formation function of VKOR, and perform functional complementation assays of E. coli strains defective in disulfide bond formation to text the disulfide bond formation function of higher plant VKOR, and study the effect of cysteines on the function of VKOR. The main results are as follows.(1) Structure analysis and transit peptide prediction of Arabidopsis VKOR. Second structure and topology of Arabidopsis VKOR were compared with other thioredoxin, which promote disulfide bond formation. The compare results indicate that the Arabidopsis VKOR contain two distinct domains, an N-terminal integral membrane domain and a C-terminal soluble DsbA-like domain. N-terminal is homologous to the microbiology VKOR, C-terminal is homologous to DsbA. So we named Arabidopsis VKOR AtVKOR-DsbA, the N-terminal named AtVKOR, and the C-terminal named AtDsbA. Transit peptide of AtVKOR was predicted online with TargetP, PredSL, and N-terminal 45 amino acid was transit peptide.(2) The clone of AtVKOR-DsbA with or without TP, AtVKOR without TP and AtDsbA in E. coli. The AtVKOR-DsbA was amplified by PCR from Arabidopsis thaliana cDNA library using gene-specific primers to construct plasmid pWL1. Plasmids pWL2 and pWL3 containing coding region of AtVKOR-DsbA but without TP were constructed using pWL1 as the template (pWL3 is His-tagged at N terminal and pWL2 is non-His-tagged). Plasmid pWL4 and pWL5 contain regions encoding AtVKOR(amino acids from 45 to 214)and AtDsbA(amino acids from 205 to 376), respectively. All the PCR products were cloned into plasmid pTrc99a.(3) We performed Motility mathod to assay the disulfide bond formation function of AtVKOR-DsbA, AtVKOR, AtDsbA. DsbA, DsbB or both were deleted E. coli strains are non-motile due to their inability to introduce a single disulfide bond critical for proper folding of the flagellar motor protein FlgI. To test the role of AtVKOR-DsbA in disulfide bond formation, we first performed functional complementation assays in E. coli Dsb null strains. The results indicated that AtVKOR-DsbA without TP could complement the defect of disulfide bond formation in Dsb null strains, and the strains showed in big colony. But individual AtVKOR domains could not replace the function of DsbB and AtDsbA domain could not substitute DsbA.(4) We performedβ-galactosidase activity determination to assay the disulfide bond formation function of AtVKOR-DsbA, AtVKOR, AtDsbA. The strains defective in disulfide bond formation containing a fusion ofβ-galactosidase to a large periplasmic domain of the cytoplasmic membrane protein MalF. If complementation can promote the formation of disulfide bonds in the exported portion of theβ-galactosidase, it would render the enzyme inactive indicated by white color, otherwise,β-galactosidase was enzymatically active, indicated by blue color. Only AtVKOR-DsbA without TP could complement the defect of disulfide bond formation in Dsb null strains, indicated by white color. This result was consistent with the Motility mathod.(5) The essential cysteins of AtVKOR-DsbA. To investigate the function of each cysteine in formation of disulfide bond, single or double mutants were obtained by Quick-change mutagenesis, and the cysteins were mutant to alanine. The results indicate that all the conserved cysteines in C-X-X-C domain and another two pairs of conserved cysteins(C109-C116, C316-C331) of AtVKOR-DsbA are essential for disulfide bond formation in E. coli, and the two non-conserved cysteines are not involved in the formation of disulfide bond.