STRUCTURAL BIOLOGY OF PROLINE CATABOLISM

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 脯氨酸脱氢酶(PROH)催化L-脯氨酸氧化生成Delta-1-吡咯啉-5-羧酸盐。PRODH表现出明显的对脯氨酸的偏好，而不是羟脯氨酸(反式-4-羟基-L-脯氨酸)作为底物，但特异性的基础尚不清楚。因此，这项研究的目标是深入了解这类酶底物专一性的结构决定因素，重点是了解PRODH如何区分两个密切相关的分子，即脯氨酸和羟脯氨酸。建立了两个大肠杆菌Puta的Proth结构域的定点突变株：Y540A和Y540S。对两个突变体进行了动力学测量。分别在1.75A、1.90A和1.85A的分辨率下测定了Y540S与羟基脯氨酸、脯氨酸及其类似物L-四氢-2-呋喃甲酸的晶体结构。Tyr540突变使羟脯氨酸的催化效率提高了3倍，而对Pro的特异性降低了20倍(Y540S)和50倍(Y540A)。结构表明，大的苯酚侧链的去除增加了底物结合口袋的体积，为羟脯氨酸的4-羟基提供了足够的空间。此外，引入的丝氨酸残基通过与4-羟基形成氢键参与了对羟基脯氨酸的识别。这一结果对于理解相关酶人羟脯氨酸脱氢酶的底物特异性具有重要意义，该酶在这个关键的活性部位用丝氨酸代替酪氨酸。动力学和结构结果表明，Tyr540是特异性的重要决定因素。在结构上，它通过与这个潜在底物的4-羟基发生碰撞而起到了负过滤羟基脯氨酸的作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Proline dehydrogenase (PRODH) catalyzes the oxidation of l-proline to Delta-1-pyrroline-5-carboxylate. PRODHs exhibit a pronounced preference for proline over hydroxyproline (trans-4-hydroxy-l-proline) as the substrate, but the basis for specificity is unknown. The goal of this study, therefore, is to gain insight into the structural determinants of substrate specificity of this class of enzyme, with a focus on understanding how PRODHs discriminate between the two closely related molecules, proline and hydroxyproline. Two site-directed mutants of the PRODH domain of Escherichia coli PutA were created: Y540A and Y540S. Kinetics measurements were performed with both mutants. Crystal structures of Y540S complexed with hydroxyproline, proline, and the proline analogue l-tetrahydro-2-furoic acid were determined at resolutions of 1.75, 1.90, and 1.85 A, respectively. Mutation of Tyr540 increases the catalytic efficiency for hydroxyproline 3-fold and decreases the specificity for proline by factors of 20 (Y540S) and 50 (Y540A). The structures show that removal of the large phenol side chain increases the volume of the substrate-binding pocket, allowing sufficient room for the 4-hydroxyl of hydroxyproline. Furthermore, the introduced serine residue participates in recognition of hydroxyproline by forming a hydrogen bond with the 4-hydroxyl. This result has implications for understanding the substrate specificity of the related enzyme human hydroxyproline dehydrogenase, which has serine in place of tyrosine at this key active site position. The kinetic and structural results suggest that Tyr540 is an important determinant of specificity. Structurally, it serves as a negative filter for hydroxyproline by clashing with the 4-hydroxyl group of this potential substrate.