STRUCTURAL BIOLOGY OF PROLINE CATABOLISM

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 脯氨酸脱氢酶（PRODH）催化l-脯氨酸氧化成δ-1-吡咯啉-5-羧酸。PRODH表现出对脯氨酸的明显偏好超过羟脯氨酸（反式-4-羟基-1-脯氨酸）作为底物，但特异性的基础是未知的。因此，本研究的目标是深入了解这类酶的底物特异性的结构决定因素，重点是了解PRODH如何区分两个密切相关的分子，脯氨酸和羟脯氨酸。产生了大肠杆菌PutA的PRODH结构域的两个定点突变体：Y 540 A和Y 540 S。用两种突变体进行动力学测量。Y 540 S与羟脯氨酸、脯氨酸和脯氨酸类似物1-四氢-2-糠酸复合的晶体结构分别在1.75、1.90和1.85 A的分辨率下测定。Tyr 540的突变使羟脯氨酸的催化效率增加3倍，并使脯氨酸的特异性降低20倍（Y 540 S）和50倍（Y 540 A）。结构表明，去除大的苯酚侧链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-羟基形成氢键参与羟脯氨酸的识别。这一结果的影响，了解相关酶的人羟脯氨酸脱氢酶，其中有丝氨酸在这个关键的活性位点位置的酪氨酸的地方的底物特异性。动力学和结构的结果表明，Tyr 540是一个重要的决定因素的特异性。在结构上，它通过与该潜在底物的4-羟基发生冲突而充当羟脯氨酸的负过滤器。

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.