Structure and Mechanism of Folate Biosynthetic Enzymes

描述(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的催化结构和分子机制，叶酸生物合成途径是开发抗菌剂的已被证实的目标途径。建议的研究是继续我们目前对6-羟甲基-7，8-二氢蝶呤焦磷酸激酶(HPPK)的研究，并扩大我们的研究范围，包括二氢蝶呤醛缩酶(DHNA)。HPPK是研究酶促焦磷酰化转移机理的良好模型。DHNA是一种独特的醛缩酶，因为它既不需要酶与底物之间形成席夫碱，也不需要金属离子来催化，而且酶还催化底物的异构化。HPPK研究的核心假设是，HPPK在其催化循环过程中经历了剧烈的构象变化，构象变化在其催化过程中起着关键作用。因此，在特定的目标1中，我们将继续探索用X射线结晶学沿着催化循环确定HPPK的结构。在具体目标2中，我们将通过时间分辨荧光能量转移(FRET)在平衡条件下甚至随着反应的进行来确定HPPK催化环的构象动力学，并通过异核核磁共振弛豫来确定其核心结构在亚纳秒到纳秒和微秒到毫秒的时间尺度上的动力学。最重要的是，在特定的目标3中，我们将通过定点突变、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。对DHNA的研究提出的主要假设是：(1)金黄色葡萄球菌和大肠杆菌的两种腺苷酶具有不同的结合/催化性质和对抑制剂的不同反应；(2)一般的酸/碱催化在这种独特的醛缩酶的催化机理中起着最关键的作用。因此，在具体目标4中，我们将通过X射线结晶学确定DHNA的结构，特别是与DHNA的Michaelis络合物最接近的新喋呤和单喋呤的配合物的结构。在具体目标5中，我们将通过定点突变、瞬时动力学pH速率剖面分析和核磁共振光谱滴定相结合的方法来鉴定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.