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

9513498安德森该项目旨在确定额外的氧化还原敏感和光调制的光合和化学合成的物种的酶。对植物酶的调查表明，令人惊讶的数量含有潜在的可氧化（或氧化）半胱氨酸残基。这些酶的活性可能是由光依赖性还原调节。在其他情况下，二硫化物可能只是起到稳定结构的作用。同源建模将被用来定位潜在的可氧化或氧化的半胱氨酸残基在这些植物酶的序列信息和模型蛋白质结构的模拟三维结构。将测试氧化剂和还原剂对粗提物中酶活性的影响，其中建议氧化还原敏感性。动物和非产氧微生物中的氧化还原敏感酶是罕见的，事实上，植物细胞质似乎比动物或微生物细胞质更具氧化性（或至少更具氧化性）。预计这些实验将证明二硫化物在植物中并不罕见，并且这些二硫化物中的许多是稳定的而不是调节的。 还原光调制在植物和蓝藻中很重要。如果植物被改造用于生产技术上重要的化合物，那么需要更多关于其特有的氧化还原生物化学的信息。拟议的实验旨在确定潜在的可氧化的半胱氨酸残基，并通过测定氧化剂和还原剂对各自酶活性的影响来测试其重要性。 在某些情况下，引入形成二硫键的Cys残基可用于稳定或控制（即当ATP或NADPH不被光合产生时关闭特定反应或途径）。拟议的实验将提供重要的洞察酶的机制，受到光调制的酶的作用，酶功能的域间运动，和潜在的光（和氧化还原）可调酶的设计用于商业用途。 ***

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. ***