（一）綠藻 (Chlorella sorokiniana T-89) glutaredoxin的晶體結構分析（二）Picrophilus torridus海藻糖合成酶的晶體結構解析與活性分析

Abstract

(一)Intracellular redox status plays an important role in cell functions, such as cell survival and proliferation. To maintain a reduced intracellular redox state, cell utilizes protein redox systems, thioredoxin (Trx) and glutaredoxin (Grx) systems. Glutaredoxins are small dithiol proteins, and can be found in many prokaryotes and eukaryotes, including humans. This system catalyzes thiol-disulfide exchange reactions or protein-mixed glutathione disulfides reduction to maintain thiol redox status of the cytosol by using glutathione (GSH) as the electron donor. To gain more knowledge on redox regulation in the single-celled green algae Chlorella, the recombinant Grx from Chlorella sorokiniana T-89 was crystallized in both oxidized and reduced states. The overall structure of Grx resembles the fold of the Trx superfamily, and Grx shares sequence similarity with human Grx2. In the active site of the oxidized Grx, Cys25 and Cys28 forms a disulfide bond. In the reduced Grx structure, glutathione makes several polar contacts with Grx. In normal state, GSH embedded in Grx catalytic active site, it may protect the catalytic activity site from free radical attacked. In the reduced status Grx would alter some side-chain conformation to accommodate GSH. We also used NCBI BLAST and the Dali the server to do a further search for similar sequences and structures of different species of Grx. We try to understand the relationship of structure and enzyme and substrate affinity between the various species This study would be very helpful for understand how Chlorella sorokiniana T89 Grx to regulate a reduced intracellular redox state to defense of oxidative stress. (二)Trehalose (trehalose) is an important economic value in the business, because it can be added to food, medicine and other products. Trehalose synthase can catalyze the conversion of maltose into trehalose in one step. Therefore, trehalose synthase is the main method to product trehalose. So far, trehalose synthase activity from many microbial species was published, but the crystal structure of the trehalose synthase was not published yet. So this experiment is to try to determine the protein structure of trehalose synthase from a thermophilic and acidophilic archaea Picrophilus torridus, and structural analysis. Now We are trying to optimizethe the crystal structure data. The other hand, the analysis of the PTTS enzyme kinetic characteristics can understand the activity of the enzyme.