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American Society for Biochemistry and Molecular Biology, Journal of Biological Chemistry, 31(285), p. 23763-23770

DOI: 10.1074/jbc.m110.115097

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Mutation Analysis of Violaxanthin De-epoxidase Identifies Substrate-binding Sites and Residues Involved in Catalysis*

This paper is made freely available by the publisher.
This paper is made freely available by the publisher.

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Abstract

Plants are able to deal with variable environmental condi- tions; when exposed to strong illumination, they safely dissipate excess energy as heat and increase their capacity for scavenging reacting oxygen species. Both these protection mechanisms involve activation of the xanthophyll cycle, in which the carote- noid violaxanthin is converted to zeaxanthin by violaxanthin de-epoxidase, using ascorbate as the source of reducing power. In this work, following determination of the three-dimensional structure of the violaxanthin de-epoxidase catalytic domain, we identified the putative binding sites for violaxanthin and ascor- bate by in silico docking. Amino acid residues lying in close con- tact with the two substrates were analyzed for their involvement in the catalytic mechanism. Experimental results supported the proposed substrate-binding sites and point to two residues, Asp-177 and Tyr-198, which are suggested to participate in the catalytic mechanism, based on complete loss of activity in mutant proteins. The role of other residues and the mechanistic similarity to aspartic proteases and epoxide hydrolases are discussed.