Vorphal María Alejandra, Bruna Carola, Wandersleben Traudy, Dagnino-Leone Jorge, Lobos-González Francisco, Uribe Elena, Martínez-Oyanedel José, Bunster Marta
Laboratorio de Biofísica Molecular, Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Barrio Universitario S/N, Casilla 160_C, Concepción, Chile.
Laboratorio de Enzimología, Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Barrio Universitario S/N, Casilla 160_C, Concepción, Chile.
Biol Res. 2017 Dec 8;50(1):39. doi: 10.1186/s40659-017-0144-5.
Ferredoxin NADP(H) oxidoreductases (EC 1.18.1.2) (FNR) are flavoenzymes present in photosynthetic organisms; they are relevant for the production of reduced donors to redox reactions, i.e. in photosynthesis, the reduction of NADP to NADPH using the electrons provided by Ferredoxin (Fd), a small FeS soluble protein acceptor of electrons from PSI in chloroplasts. In rhodophyta no information about this system has been reported, this work is a contribution to the molecular and functional characterization of FNR from Gracilaria chilensis, also providing a structural analysis of the complex FNR/Fd.
The biochemical and kinetic characterization of FNR was performed from the enzyme purified from phycobilisomes enriched fractions. The sequence of the gene that codifies for the enzyme, was obtained using primers designed by comparison with sequences of Synechocystis and EST from Gracilaria. 5'RACE was used to confirm the absence of a CpcD domain in FNRPBS of Gracilaria chilensis. A three dimensional model for FNR and Fd, was built by comparative modeling and a model for the complex FNR: Fd by docking.
The kinetic analysis shows K of 12.5 M and a k of 86 s, data consistent with the parameters determined for the enzyme purified from a soluble extract. The sequence for FNR was obtained and translated to a protein of 33646 Da. A FAD and a NADP+ binding domain were clearly identified by sequence analysis as well as a chloroplast signal sequence. Phycobilisome binding domain, present in some cyanobacteria was absent. Transcriptome analysis of Gch revealed the presence of two Fd; FdL and FdS , sharing the motif CX5CX2CX29X. The analysis indicated that the most probable partner for FNR is FdS.
The interaction model produced, was consistent with functional properties reported for FNR in plants leaves, and opens the possibilities for research in other rhodophyta of commercial interest.
铁氧化还原蛋白NADP(H)氧化还原酶（EC 1.18.1.2）（FNR）是光合生物中存在的黄素酶；它们与氧化还原反应中还原供体的产生相关，即在光合作用中，利用铁氧化还原蛋白（Fd，一种来自叶绿体中光系统I的小的FeS可溶性电子受体）提供的电子将NADP还原为NADPH。在红藻中尚未有关于该系统的信息报道，这项工作有助于对智利江蓠FNR进行分子和功能表征，同时也对FNR/Fd复合物进行结构分析。
FNR的生化和动力学表征是通过从富含藻胆体的级分中纯化的酶进行的。通过与集胞藻序列和智利江蓠EST序列比较设计引物，获得编码该酶的基因序列。使用5'RACE来确认智利江蓠FNRPBS中不存在CpcD结构域。通过比较建模构建FNR和Fd的三维模型，并通过对接构建FNR:Fd复合物的模型。
动力学分析显示K为12.5 μM，k为86 s，该数据与从可溶性提取物中纯化的酶所确定的参数一致。获得了FNR的序列，并将其翻译成一个33646 Da的蛋白质。通过序列分析清楚地鉴定出一个FAD和一个NADP+结合结构域以及一个叶绿体信号序列。在一些蓝细菌中存在的藻胆体结合结构域不存在。对Gch的转录组分析揭示了存在两种Fd；FdL和FdS，它们共享基序CX5CX2CX29X。分析表明FNR最可能的伙伴是FdS。
所产生的相互作用模型与植物叶片中报道的FNR功能特性一致，并为其他具有商业价值的红藻的研究开辟了可能性。
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