Covalent Phosphorus Residues in Flavoproteins

拟议的工作将集中在两种蛋白质中与蛋白质结合的共价磷残基的结构和功能：棕色固氮菌(菌株OP)黄毒素和牛乳黄嘌呤氧化酶。在黄还蛋白的情况下，已经确定了丝基和苏氨基残基之间的一种新的磷酸二酯交联物。过去项目期的工作表明，这种交联物似乎具有结构功能，克隆黄毒素基因并在大肠杆菌中表达可以形成没有这种共价磷残基的黄毒素。PI计划将这些研究扩展到1)鉴定磷酸桥联的丝氨基和苏氨基残基的序列位置，2)检查“去磷”克隆的黄还蛋白与磷酸野生型的比较物理和化学性质，3)开始研究负责将磷酸二酯键结合到蛋白质中的酶系统，4)启动使用定点突变作为蛋白质结构和功能探针的研究，5)鉴定在棕色固氮菌(菌株478)黄毒素中可能存在的非磷酸交联物，以及可能存在于从其他生物分离的黄毒素中，和6)制备针对含有磷酸二酯连接的合成肽的多克隆抗体，以用作探针，以测试这种连接是否发生在其他生物的蛋白质中。这个实验室以前的工作(由NSF支持)已经表明，牛乳黄嘌呤氧化酶在该酶的活性中心钼中心附近含有一个磷酸丝氨酸残基。初步的31P核磁共振和质谱学数据表明，底物周转导致溶剂氧进入这个磷残留物。有人建议扩大这些发现的范围，以确定该磷酸丝氨酸残基是否是参与黄嘌呤氧化机制的活性部位亲核试剂。~(32)P共价掺入相关酶的证据：鸡肝黄嘌呤脱氢酶和兔肝醛氧化酶将在~(32)PI存在下进行细胞生长，并用抗体检测合成的黄嘌呤脱氢酶和醛氧化酶是否也含有共价结合的磷残基。从这个实验室的Endor数据和Bray最近发表的Endor数据中，有证据表明黄嘌呤氧化酶的功能性钼中心有一个磷配体。将进行进一步的实验，以进一步确定该信号是否确实是由于磷以及该磷是否是由于磷酸丝氨酸残基。这些研究的结果应该会扩大我们对磷残基在蛋白质结构和功能中的作用的理解，并可能与生物化学中的一系列问题相关。

The proposed work will focus on the structure and function of protein-bound covalent phosophorus residues in two proteins: Azotobacter vinelandii (strain OP) flavodoxin and bovine milk xanthine oxidase. In the case of flavodoxin, a novel phosphodiester crosslink between a seryl and a threonyl residue has been identified. Work over the past project period has shown this crosslink appears to have a structural function and that cloning the flavodoxin gene and expressing it in E. coli results in formation of the flavodoxin without this covalent phosphorus residue. The PI plans to extend these studies to 1) identify the sequence location of the phosphate-bridged seryl and threonyl residues, 2) examine the comparative physical and chemical properties of the "dephospho" cloned flavodoxin with that of the phospho wild-type form, 3) initiate studies to characterize the enzyme system responsible for incorporation of the phosphodiester linkage into the protein, 4) initiate studies using site-directed mutagenesis as a probe of protein structure and function, 5) identify non-phospho crosslinks suggested to occur in Azotobacter vinelandii (strain 478) flavodoxin and maybe present in flavodoxin isolated from other organisms, and 6) prepare a polyclonal antibody against a synthetic peptide containing a ser-thr phosphodiester linkage to use as a probe to test whether this linkage occurs in proteins in other organisms. Previous work in this laboratory (supported by NSF) has shown bovine milk xanthine oxidase to contain a phosphoseryl residue near the active site Mo center of the enzyme. Preliminary 31P NMR and mass spectroscopic data suggest substrate turnover results in the incorporation of solvent oxygen into this phosphorus residue. Work is proposed to extend these findings to determine if this phosphoseryl residue is the active site nucleophile involved in the mechanism of xanthine oxidation. Evidence for covalent 32P incorporation in the related enzymes: chicken liver xanthine dehydrogenase and rabbit liver aldehyde oxidase will be obtained by cell growth in the presence of 32Pi and using antibodies to probe whether synthesized xanthine dehydrogenase and aldehyde oxidase also contain covalently-bound phosphorus residues. From this laboratory ENDOR data and recent ENDOR data published by Bray; evidence is presented to suggest a phosphorus ligand to the functional Mo center of xanthine oxidase. Further experiments will be done to further determine whether this signal is indeed due to phosphorus and whether that phosphorus is due to the phosphoseryl residue. The results of these studies should expand our understanding of the role of phospho residues in protein structure and function and may have relevance to a wide range of problems in biochemistry.