A new catalytic mechanism of bacterial ferredoxin-NADP+ reductases due to a particular NADP+ binding mode
A new catalytic mechanism of bacterial ferredoxin-NADP+ reductases due to a particular NADP+ binding mode
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DOI：
10.1002/pro.4166
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发表时间：
2021-08-21
期刊：
影响因子：
8
通讯作者：
Catalano-Dupuy, Daniela L.
中科院分区：
文献类型：
--
作者：
Monchietti, Paula;Lopez Rivero, Arleth S.;Catalano-Dupuy, Daniela L.
铁氧还蛋白-NADP（+）还原酶（FNR）是广泛存在的参与氧化还原代谢的黄素酶。FNR催化NADP（H）与铁氧还蛋白或黄素氧还蛋白之间的可逆电子转移。它们被分类为植物型和植物型FNR。植物型FNR分为质体类和细菌类。质体FNR的周转率比细菌酶高20至100倍，这些差异与它们的生理功能有关。我们证明了纯化的大肠杆菌FPR（EcFPR）含有紧密结合的NADP（+），这在质体型FNR中不存在。EcFPR的三维结构证明NADP（+）与三个碱基（R144、R174和R184）相互作用，可以产生非常高的亲和力和结构化位点。这些酶在其他细菌FNR中是保守的，但在质体酶中不是。我们用豌豆FNR（PsFNR）中类似位置上存在的残基交叉取代EcFPR丝氨酸，并用PsFNR中的丝氨酸取代这些氨基酸。我们通过结构、动力学和稳定性研究分析了所有蛋白质。我们发现EcFPR突变体不含结合的NADP（+），并显示该核苷酸的Km增加。NADP（+）可抑制EcFPR活性，但这种抑制作用随底物浓度的降低而消失。烟酰胺部分的NADP（+）类似物对EcFPR产生激活作用并促进NADP（+）释放。我们的研究结果为细菌FNR中NADP（+）结合和催化的新模型提供了证据，我们认为这种紧密的NADP（+）结合构成了细菌FNR参与氧化还原稳态的重要催化和调节机制。
Ferredoxin-NADP(+) reductases (FNRs) are ubiquitous flavoenzymes involved in redox metabolisms. FNRs catalyze the reversible electron transfer between NADP(H) and ferredoxin or flavodoxin. They are classified as plant- and mitochondrial-type FNR. Plant-type FNRs are divided into plastidic and bacterial classes. The plastidic FNRs show turnover numbers between 20 and 100 times higher than bacterial enzymes and these differences have been related to their physiological functions. We demonstrated that purified Escherichia coli FPR (EcFPR) contains tightly bound NADP(+), which does not occur in plastidic type FNRs. The three-dimensional structure of EcFPR evidenced that NADP(+) interacts with three arginines (R144, R174, and R184) which could generate a very high affinity and structured site. These arginines are conserved in other bacterial FNRs but not in the plastidic enzymes. We have cross-substituted EcFPR arginines with residues present in analogous positions in the Pisum sativum FNR (PsFNR) and replaced these amino acids by arginines in PsFNR. We analyzed all proteins by structural, kinetic, and stability studies. We found that EcFPR mutants do not contain bound NADP(+) and showed increased K-m for this nucleotide. The EcFPR activity was inhibited by NADP(+) but this behavior disappeared as arginines were removed. A NADP(+) analog of the nicotinamide portion produced an activating effect on EcFPR and promoted the NADP(+) release. Our results give evidence for a new model of NADP(+) binding and catalysis in bacterial FNRs.We propose that this tight NADP(+) binding constitutes an essential catalytic and regulatory mechanism of bacterial FNRs involved in redox homeostasis.