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

该项目旨在确定光合和化学合成物种中额外的氧化还原敏感和光调节酶。一项对植物酶的调查表明，惊人的数量含有潜在的可氧化（或氧化）半胱氨酸残基。其中一些酶的活性可能受光依赖性还原的调节。在其他情况下，二硫化物可能只是起到稳定结构的作用。同源性建模将用于在这些植物酶的模拟三维结构中定位潜在的可氧化或被氧化的半胱氨酸残基，这些酶的序列信息和模型蛋白质结构都是可用的。氧化还原剂和还原剂对粗提取物中酶活性的影响将在提出氧化还原敏感性的情况下进行测试。动物和非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有氧化性（或至少在某种程度上更具有氧化性）。预计这些实验将证明，二硫化物在植物中并不罕见，而且这些二硫化物中的许多是稳定的，而不是调节的。还原性光调制在植物和蓝藻中很重要。如果要对植物进行改造以生产技术上重要的化合物，就需要更多关于它们特有的氧化还原生物化学的信息。提出的实验旨在通过分析氧化剂和还原剂对相应酶活性的影响来确定潜在可氧化的胱氨酸残基并测试其重要性。在某些情况下，引入二硫化物形成的Cys残基可以用于稳定或控制（即当ATP或NADPH不进行光合作用时关闭特定的反应或途径）。提出的实验将对酶的机制、受光调制影响的酶的作用、酶功能的域间运动以及设计用于商业用途的光（和氧化还原）可调节酶的潜力提供重要的见解。＊＊＊

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. ***