Mechanism of dihydroneopterin aldolase: functional roles of the conserved active site glutamate and lysine residues.
Mechanism of dihydroneopterin aldolase: functional roles of the conserved active site glutamate and lysine residues.
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二氢新蝶呤醛缩酶的机制：保守活性位点谷氨酸和赖氨酸残基的功能作用。
DOI：
10.1021/bi060949j
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发表时间：
2006
期刊：
影响因子：
2.9
通讯作者：
Yan,Honggao
中科院分区：
文献类型：
--
作者：
Wang,Yi;Li,Yue;Yan,Honggao
二氢新蝶呤醛缩酶 (DHNA) 在叶酸生物合成途径中催化 7,8-二氢新蝶呤 (DHNP) 转化为 6-羟甲基-7,8-二氢蝶呤 (HP)。金黄色葡萄球菌DHNA（SaDHNA）活性位点有4个保守活性位点残基E22、Y54、E74和K100，分别对应于大肠杆菌DHNA（EcDHNA）中的E21、Y53、E73和K98。在这项工作中，通过定点诱变研究了保守的谷氨酸和赖氨酸残基的功能作用。 SaDHNA 的 E22 和 E74 以及 EcDHNA 的 E21、E73 和 K98 被替换为丙氨酸。 SaDHNA 的 K100 被丙氨酸和谷氨酰胺取代。通过平衡结合、停流结合和稳态动力学分析来表征突变蛋白。对于 SaDHNA，除了 E74A 之外，没有任何突变会导致酶对底物或产物类似物的亲和力或速率常数发生显着变化。 SaE74A 的 Kd 值估计>3000 μM，表明突变体的 Kd 值至少是野生型酶的 100 倍。对于 EcDHNA，相对于野生型酶，E73A 突变使底物或产物类似物新蝶呤 (MP)、单喋呤 (NP) 和 6-羟基蝶呤 (HPO) 的 Kd 值分别增加了 340、160 和 5600 倍。 K98A 突变使 NP、MP 和 HPO 的 Kd 值分别增加 14、3.6 和 230 倍。 E21A突变使NP和HPO的Kd值分别增加2.2和42倍，但使MP的Kd值降低3.3倍。 E22 (E21) 和 K100 (K98) 突变使 kcat 值降低了 1.3−2 × 104 倍。E74 (E73) 突变使 kcat 值降低了 ∼10 倍。结果表明，SaDHNA 的 E74 和 EcDHNA 的 E73 对于底物结合很重要，但它们在催化中的作用较小。相比之下，SaDHNA 的 E22 和 K100 对于催化很重要，但它们在底物结合中的作用较小。另一方面，EcDHNA 的 E21 和 K98 对于底物结合和催化都很重要。
Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) in the folate biosynthetic pathway. There are four conserved active site residues at the active site, E22, Y54, E74, and K100 inStaphylococcus aureusDHNA (SaDHNA), corresponding to E21, Y53, E73, and K98, respectively, inEscherichia coliDHNA (EcDHNA). The functional roles of the conserved glutamate and lysine residues have been investigated by site-directed mutagenesis in this work. E22 and E74 of SaDHNA and E21, E73, and K98 of EcDHNA were replaced with alanine. K100 of SaDHNA was replaced with alanine and glutamine. The mutant proteins were characterized by equilibrium binding, stopped-flow binding, and steady-state kinetic analyses. For SaDHNA, none of the mutations except E74A caused dramatic changes in the affinities of the enzyme for the substrate or product analogues or the rate constants. TheKdvalues for SaE74A were estimated to be >3000 μM, suggesting that theKdvalues of the mutant are at least 100 times those of the wild-type enzyme. For EcDHNA, the E73A mutation increased theKdvalues for the substrate or product analogues neopterin (MP), monapterin (NP), and 6-hydroxypterin (HPO) by factors of 340, 160, and 5600, respectively, relative to those of the wild-type enzyme. The K98A mutation increased theKdvalues for NP, MP, and HPO by factors of 14, 3.6, and 230, respectively. The E21A mutation increased theKdvalues for NP and HPO by factors of 2.2 and 42, respectively, but decreased theKdvalue for MP by a factor of 3.3. The E22 (E21) and K100 (K98) mutations decreased thekcatvalues by factors of 1.3−2 × 104. The E74 (E73) mutation decreased in thekcatvalues by factors of ∼10. The results suggested that E74 of SaDHNA and E73 of EcDHNA are important for substrate binding, but their roles in catalysis are minor. In contrast, E22 and K100 of SaDHNA are important for catalysis, but their roles in substrate binding are minor. On the other hand, E21 and K98 of EcDHNA are important for both substrate binding and catalysis.