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; 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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 脯氨酸脱氢酶（PODH）催化L-丙啉氧化为Delta-1-吡咯酸5-羧酸盐的氧化。 Prodhs表现出对脯氨酸的明显偏好而不是羟基（Trans-4-羟基L-丙啉）作为底物，但特异性的基础尚不清楚。因此，这项研究的目的是深入了解该类酶的底物特异性的结构决定因素，重点是理解prodhs如何区分两个密切相关的分子，脯氨酸和羟基丙烯。创建了大肠杆菌PUTA的两个位置定向突变体：Y540A和Y540S。用两个突变体进行动力学测量。 Y540的晶体结构分别在1.75、1.90和1.85 a的分辨率下确定了与羟丙基，脯氨酸和脯氨酸类似物L-tetrahydro-2-氟酸的晶体结构。 Tyr540的突变提高了3倍羟基的催化效率，并通过20（Y540）和50（Y540A）的因素降低了脯氨酸的特异性。该结构表明，去除大苯酚侧链会增加底物结合口袋的体积，从而为羟基普罗林的4-羟基提供足够的空间。此外，引入的丝氨酸残基通过与4-羟基形成氢键来参与识别羟基普罗林。该结果对理解相关酶的人羟基脱氢酶的底物特异性具有影响，该酶在此关键的活性位置位于该关键的活性位置，它代替酪氨酸代替酪氨酸。动力学和结构结果表明Tyr540是特异性的重要决定因素。从结构上讲，它通过与该电势底物的4-羟基相冲突来充当羟基丙烯酸酯的负滤波器。