Covalent Phosphorus Residues in Flavoproteins

拟议的工作将侧重于 蛋白质结合的共价磷残基在两个 蛋白质：棕色固氮菌（菌株OP）黄氧还蛋白和 牛乳黄嘌呤氧化酶 在黄素氧还蛋白的情况下， 丝氨酰基和苏氨酰基残基之间磷酸二酯交联 已经被确认了 在过去的项目期间， 显示出这种交联剂似乎具有结构功能， 克隆黄素氧还蛋白基因并在E.杆菌 导致形成没有这种共价键的黄素氧还蛋白 磷渣 PI计划将这些研究扩展至1） 鉴定磷酸桥连丝氨酰基的序列位置， 苏氨酰残基，2）检查比较物理和 “去磷酸”克隆的黄素氧还蛋白的化学性质， 磷酸化野生型形式，3）启动研究， 表征负责掺入的酶系统 将磷酸二酯连接到蛋白质中，4）启动研究 利用定点突变作为蛋白质结构的探针 和功能，5）确定非磷酸交联建议， 存在于棕色固氮菌（菌株478）中， 可能存在于从其他生物体分离的黄素氧还蛋白中， 6)制备针对合成肽的多克隆抗体 含有Ser-Thr磷酸二酯键，用作探针， 测试这种连接是否发生在其他生物的蛋白质中。 该实验室以前的工作（由NSF支持）表明， 牛乳黄嘌呤氧化酶含有磷酸丝氨酰残基 在酶的活性位点Mo中心附近。 初步 31 P NMR和质谱数据表明底物 周转导致溶剂氧掺入其中， 磷渣 建议开展工作，将这些发现扩展到 确定这个磷酸丝氨酰残基是否是活性位点 亲核试剂参与黄嘌呤的氧化机制。 相关酶中共价32 P掺入的证据： 鸡肝黄嘌呤脱氢酶和兔肝醛 将通过在32 Pi存在下的细胞生长获得氧化酶 并使用抗体来探测合成的黄嘌呤 脱氢酶和醛氧化酶也含有共价结合的 磷残留物 从这个实验室ENDOR数据和最近的 ENDOR数据由布雷发表;证据表明， 黄嘌呤功能性钼中心的磷配体 氧化酶。 将进行进一步的实验以进一步确定 这个信号是否真的是由于磷， 磷是由于磷酸丝氨酰残基。 的结果 这些研究应该扩大我们对 蛋白质结构和功能中的磷酸残基， 与生物化学中广泛的问题相关。

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.