Identification of Intra-Cellular Enzymes which Contain Disulfide Bonds in Plants

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9513498 Anderson This project seeks to identify additional redox-sensitive and light-modulated enzymes in photosynthetic and chemosynthetic species. A survey of plant enzymes indicates that a surprising number contain potentially oxidizable (or oxidized) cysteine residues. The activity of some of these enzymes is probably regulated by light-dependent reduction. In others, disulfides may simply act to stabilize structure. Homology modeling will be used to locate potentially oxidizable or oxidized cysteine residues in the simulated three-dimensional structures of those plant enzymes for which both sequence information and a model protein structure is available. The effect of oxidizing and reducing agents on the activity of the enzymes in crude extracts will be tested where redox-sensitivity is suggested. Redox-sensitive enzymes in animals and non-oxygenic microbes are rare, and in fact the plant cytosol appears to be much more oxidizing (or at least somewhat more oxidizing) than animal or microbial cytosol. It is expected that these experiments will demonstrate that disulfides are not unusual in plants and that many of these disulfides are stabilizing rather than regulatory. %%% Reductive light modulation is important in plants and cyanobacteria. If plants are to be engineered for the production of technologically important compounds, more information about their peculiar redox biochemistry is needed. The proposed experiments aim to identify potentially oxidizable Cys residues and to test their importance by assaying the effect of oxidizing and reducing agents on the activity of the respective enzymes. In some cases introduction of disulfide-forming Cys residues could be useful either for stabilization or for control (i.e. turning specifia reactions or pathways off when ATP or NADPH are not being photosynthetically generated). The proposed experiments will give important insight into enzyme mechanisms, the action of the enzymes which are subject to light modulation, interdomain movements in enzyme function, and the potential for the design of light (and redox) modulatable enzymes for commercial use. ***