A Single Tyrosine Hydroxyl Group Almost Entirely Controls the NADPH Specificity of Plasmodium falciparum Ferredoxin-NADP+ Reductase
A Single Tyrosine Hydroxyl Group Almost Entirely Controls the NADPH Specificity of Plasmodium falciparum Ferredoxin-NADP+ Reductase
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DOI：
10.1021/bi300078p
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发表时间：
2012-05-08
期刊：
影响因子：
2.9
通讯作者：
Aliverti, Alessandro
中科院分区：
文献类型：
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作者：
Baroni, Sara;Pandini, Vittorio;Aliverti, Alessandro
恶性疟原虫铁氧还蛋白-NADP（+）还原酶（FNR）是一种含有FAD的酶，除了是新型抗疟药物的有希望的靶标之外，还代表了植物型FNR的优良模型。FNR的辅因子特异性取决于NADPH和NADH的k（cat）和K-m值的差异。在这里，我们报告了恶性疟原虫FNR的保守Y258的羟基基团的缺失，其与NADPH的2 '-磷酸基团相互作用，选择性地将NADPH依赖性反应的k（cat）降低2倍，以匹配NADH依赖性反应的k（cat）。快速反应动力学、NADP（+）活性位点滴定和厌氧光还原实验表明，这种效应可能是结合NADPH的催化活性构象不稳定的结果。此外，由于Y258 F替换增加了NADPH的K-m 4倍，降低了NADH的K-m 3倍，导致酶区分辅酶的能力从70倍下降到仅1.5倍。Y258 F变化的影响不受H286 Q突变的影响，已知H286 Q突变会增强酶的催化活性。我们的数据突出了Y258羟基在决定恶性疟原虫FNR的辅酶特异性中所起的主要作用。从工程化植物型FNR的动力学性质的一般观点来看，虽然恶性疟原虫FNR比其同系物不那么严格地依赖NADPH，但本文报道的几乎完全消除辅酶选择性以前从未通过单一突变实现。
Plasmodium falciparum ferredoxin-NADP(+) reductase (FNR) is a FAD-containing enzyme that, in addition to be a promising target of novel antimalarial drugs, represents an excellent model of plant-type FNRs, The cofactor specificity of FNRs depends on differences in both k(cat) and K-m values for NADPH and NADH. Here, we report that deletion of the hydroxyl group of the conserved Y258 of P. falciparum FNR, which interacts with the 2'-phosphate group of NADPH, selectively decreased the k(cat) of the NADPH-dependent reaction by a factor of 2 to match that of the NADH-dependent one. Rapid-reaction kinetics, active-site titrations with NADP(+), and anaerobic photoreduction experiments indicated that this effect may be the consequence of destabilization of the catalytically competent conformation of bound NADPH. Moreover, because the Y258F replacement increased the K-m for NADPH 4-fold and decreased that for NADH 3-fold, it led to a drop in the ability of the enzyme to discriminate between the coenzymes from 70- to just 1.5-fold. The impact of the Y258F change was not affected by the presence of the H286Q mutation, which is known to enhance the catalytic activity of the enzyme. Our data highlight the major role played by the Y258 hydroxyl group in determining the coenzyme specificity of P. falciparum FNR. From the general standpoint of engineering the kinetic properties of plant-type FNRs, although P. falciparum FNR is less strictly NADPH-dependent than its homologues, the almost complete abolishment of coenzyme selectivity reported here has never been accomplished before through a single mutation.