Structure and Mechanism of Folate Biosynthetic Enzymes

描述（由申请人提供）：该项目的长期目标是确定叶酸生物合成途径中酶催化的结构和分子机制，叶酸生物合成途径是开发抗菌剂的经证实的靶向途径。本研究是对6-羟甲基-7，8-二氢蝶呤焦磷酸激酶（HPPK）的进一步研究，并将其应用于二氢新蝶呤醛缩酶（DHNA）的研究。HPPK是研究酶促焦磷酸转移机理的理想模型。DHNA是一种独特的醛缩酶，因为它既不需要在酶与其底物之间形成席夫碱，也不需要金属离子进行催化，并且酶还催化其底物的差向异构化。HPPK拟议研究背后的核心假设是，基于上一个资助期获得的结果，即HPPK在其催化周期中经历了剧烈的构象变化，而构象变化在其催化过程中起着关键作用。因此，在具体目标1中，我们将通过X射线晶体学继续探索沿着催化循环的HPPK结构测定。在具体目标2中，我们将通过时间分辨荧光能量转移（FRET）在平衡条件下确定HPPK催化环的构象动力学，甚至随着反应的进行，以及通过在亚纳秒至纳秒和微秒至毫秒时间尺度上的异频NMR弛豫确定其核心结构的动力学。最重要的是，在具体目标3中，我们将通过定点诱变，生化分析（特别是瞬时动力学分析）和生物物理方法将HPPK的结构和构象动力学与其催化作用相关联。DHNA研究的主要假设是：（1）来自金黄色葡萄球菌和大肠杆菌的两种Adlolase; coil具有不同的结合/催化性质和对抑制剂的不同响应;（2）一般的酸/碱催化在该独特的醛缩酶的催化机制中起着最关键的作用。因此，在具体目标4中，我们将通过X射线晶体学确定DHNA的结构，特别是与新蝶呤和异蝶呤的复合物的结构，它们是DHNA的米氏复合物的最接近的模拟物。在具体目标5中，我们将通过定点诱变、瞬时动力学pH速率曲线分析和NMR光谱滴定的组合来鉴定DHNA中参与一般酸/碱催化的残基。

DESCRIPTION (provided by applicant): The long-term goal of the project is to determine the structures and molecular mechanisms of catalysis for enzymes in the folate biosynthetic pathway, a proven target pathway for developing antimicrobial agents. The proposed research is to continue our current study on 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), and to expand our research to include dihydroneopterin aldolase (DHNA). HPPK is an excellent model for studying mechanism of enzymatic pyrophosphoryl transfer. DHNA is a unique aldolase because it requires neither the formation of a Schiff base between the enzyme and its substrate nor metal ions for catalysis, and the enzyme also catalyzes the epimerization of its substrate. The central hypothesis behind the proposed research on HPPK, which is based on the results obtained in the previous funding period, is that HPPK undergoes dramatic conformational changes during its catalytic cycle and the conformational changes play critical roles in its catalysis. Thus, in Specific Aim 1, we will continue our quest for structure determination of HPPK along the catalytic cycle by X-ray crystallography. In Specific Aim 2, we will determine the conformational dynamics of the catalytic loops of HPPK by time-resolved fluorescence energy transfer (FRET) at equilibrium conditions and even as the reaction progresses and the dynamics of its core structure by heteronuclear NMR relaxation at the sub-nanosecond to nanosecond and microsecond to millisecond time scales. Most importantly, in Specific Aim 3, we will correlate the structure and conformational dynamics of HPPK with its catalysis by site-directed mutagenesis, biochemical analysis (particularly transient kinetic analysis), and biophysical methods. The main hypotheses behind the proposed research on DHNA are that (1) the two adlolases from Staphylococcus aureus and E. coil have different binding/catalytic properties and distinct responses to inhibitors and (2) general acid/base catalysis plays a most critical role in the catalytic mechanism of this unique aldolase. Thus, in Specific Aim 4, we will determine the structures of DHNA by X-ray crystallography, particularly the structures of the complexes with neopterin and monopterin, the closest mimics of the Michaelis complexes of DHNA. In Specific Aim 5, we will identify residues involved in general acid/base catalysis in DHNA by a combination of site-directed mutagenesis, transient kinetic pH-rate profile analysis, and NMR spectroscopic titration.