Key Amino Acids of Arabidopsis VKOR in the Activity of Phylloquinone Reduction and Disulfide Bond Formation
2014
Many proteins in chloroplast are regulated through the disulfide bond/thiol transformation to realize
their activities. A homologue of VKOR (Vitamin K epoxide reductase) in Arabidopsis chloroplast is
found to catalyze the disulfide bond formation in vivo and to mediate the specific phylloquinone reduction
in vitro. It is also called LTO1 (Lumen Thiol Oxidoreductase 1). Investigations about functions and essential
amino acid residues of AtVKOR have important theoretical significance to clarify the chloroplast redox
regulation mechanism. In this study, several amino acids in the VKOR domain of AtVKOR were identified
to be involved in binding of phylloquinone. Site-directed mutagenesis was used to study the function of
these positions. The results suggested that residues Ser77, Leu87, Phe137 and Leu141 were quite important
in the binding and catalyzing the reduction of phylloquinone. These residues were also involved in the electron transferring
and disulfide bond formation of substrate proteins by motility assays in vivo, suggesting that the binding of phylloquinone
not only affected the delivery of electrons to phylloquinone but also affected the whole electron transfer process.
The conserved cysteines in the AtVKOR domain also played critical roles in phylloquinone reduction. When each of the
four conserved cysteines was mutated to alanine, the mutants lost reduction activity entirely, suggesting that the four conserved
cysteines played crucial roles in the electron transfer process.
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