Y XAA 3K PROTEIN FAMILY--STRUCTURE AND MECHANISM

最近的结构研究表明，二氢蝶呤还原酶(DHPR)是 二核苷酸大家族的成员，需要酶，包括 短链脱氢酶和碳水化合物差向异构酶。已知的 不同基团的成员具有不到20%的序列一致性 以同样的方式折叠。每种蛋白质都含有一个富含甘氨酸的β-α-β-折叠 可以容纳二核苷酸辅因子的AMP成分，并且 每个序列在活性中心附近都含有一个Y(Xaa)/3K基序。在……里面 事实上，在整个生命周期中，总共至少有五种保守的氨基酸 家庭，似乎对创造一种环境至关重要 可能会发生氢化物转移。催化了三个不同的反应； 还原、氧化和异构化。在每一种情况下，都是一个极化的替身 生成或还原C=N或C=O等键。使用特定站点- 定向诱变和X射线结晶学，这项提议旨在 确定哪些结构特征是允许这样一组 顺序不同的蛋白质以形成明显相似的结构 然而，保持足够的认同感，可能会出现三种不同的反应。 每种需要酶的二核苷酸共同的唯一假说 机制是酪氨酸/赖氨酸基序可能是一种新的方法 促进与酪氨酸酚基的质子交换(pk约 10)。旨在证实这一点的特定突变和动力学实验 描述了DHPR和2，3-二氢-2，3-二氢-2，3-环己烷的概念 要提纯的二羟基苯甲酸脱氢酶(DDBDH)， 结晶的和结构表征的。据预计， 实验结果将证明，结构折叠模式 确保保守氨基酸保留特征功能是 在寻找蛋白质时，与序列同等或更重要 行为模式。 作为调查的必然结果，它还打算推导出 编码抗药性蛋白的利什曼原虫LTDH的结构 显示出与上述酶家族不同寻常的序列相似性。

Recent structural studies reveal that dihydropteridine reductase (DHPR) is a member of a large family of dinucleotide requiring enzymes comprising the short-chain dehydrogenases and carbohydrate epimerases. The known members of the differing groups have less than 20% sequence identity yet fold similarly. Each protein contains a glycine rich beta-alpha-beta-fold that can accommodate the AMP component of the dinucleotide cofactor and each contains a Y(Xaa)/3K motif in the vicinity of the active site. In fact, a total of at least five conserved amino acids occur throughout the family and appear essential to the creation of an environment in which a hydride transfer can occur. Three distinct reactions are catalyzed; reduction; oxidation, and epimerisation. In each case a polarized double bond such as C=N or C=O is created or reduced. Using specific site- directed mutagenesis and X-ray crystallography, this proposal intends to determine what structural features are necessary to allow such a group of sequentially disparate proteins to formulate apparently similar structures yet retain sufficient identity that three differing reactions can occur. A unique hypothesis common to each of the dinucleotide requiring enzyme mechanisms is that the tyrosine/lysine motif could be a novel method for facilitating proton exchange with the tyrosine phenolic group (pK about 10). Specific mutational and kinetic experiments that aim to confirm this concept are described for both DHPR and the 2,3-dihydro-2,3- dihydroxybenzoate dehydrogenase (DDBDH), which is to be purified, crystallized and structurally characterized. It is anticipated that the experimental results will demonstrate that structural folding patterns that ensure conserved amino acids retain a characteristic function are equally or more important than sequence when looking for protein behavioral patterns. As a corollary to the investigation it is also intended to derive the structure of the Leishmania ltdh encoding resistance protein, as it demonstrates unusual sequence similarities to the above family of enzymes.