STRUCTURAL BIOLOGY OF PROLINE CATABOLISM

脯氨酸分解代谢的结构生物学

• 批准号: 7955213
• 负责人: JOHN J TANNER
• 金额: $ 0.13万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955213
• 关键词: Acids; Active Sites; Binding; Catabolism; Complex; Computer Retrieval of Information on Scientific Projects Database; Enzymes; Escherichia coli; Excision; Exhibits; Funding; Goals; Grant; Human; Hydrogen Bonding; Hydroxyl Radical; Hydroxyproline; Institution; Kinetics; Measurement; Mutation; Oxidoreductase; Phenols; Positioning Attribute; Proline; Proline Dehydrogenase; Research; Research Personnel; Resolution; Resources; Serine; Side; Site; Source; Specificity; Structure; Substrate Specificity; Tyrosine; United States National Institutes of Health; analog; base; carboxylate; hydroxyl group; insight; mutant; oxidation; preference; pyrroline; structural biology

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 脯氨酸脱氢酶 (PRODH) 催化 L-脯氨酸氧化为 Delta-1-吡咯啉-5-羧酸盐。 PRODH 对脯氨酸表现出明显的偏好，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，本研究的目标是深入了解此类酶底物特异性的结构决定因素，重点是了解 PRODH 如何区分脯氨酸和羟脯氨酸这两种密切相关的分子。创建了大肠杆菌 PutA 的 PRODH 结构域的两个定点突变体：Y540A 和 Y540S。对两种突变体进行了动力学测量。分别以 1.75、1.90 和 1.85 A 的分辨率测定了与羟脯氨酸、脯氨酸和脯氨酸类似物 L-四氢-2-糠酸复合的 Y540S 的晶体结构。 Tyr540 的突变使羟脯氨酸的催化效率提高了 3 倍，并使脯氨酸的特异性降低了 20 倍 (Y540S) 和 50 倍 (Y540A)。结构表明，去除大的苯酚侧链会增加底物结合袋的体积，为羟脯氨酸的 4-羟基留出足够的空间。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，该酶在这个关键活性位点位置用丝氨酸代替了酪氨酸。动力学和结构结果表明 Tyr540 是特异性的重要决定因素。从结构上讲，它通过与潜在底物的 4-羟基发生冲突，充当羟脯氨酸的负过滤器。