Biochemical basis for redox regulation of chloroplast-localized phosphofructokinase from Arabidopsis thaliana.
Biochemical basis for redox regulation of chloroplast-localized phosphofructokinase from Arabidopsis thaliana.
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DOI：
10.1093/pcp/pcaa174
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发表时间：
2021-01
影响因子：
4.9
通讯作者：
Keisuke Yoshida;T. Hisabori
中科院分区：
文献类型：
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作者：
Keisuke Yoshida;T. Hisabori
植物叶绿体中的多种蛋白质受到基于硫醇的氧化还原调控，从而实现叶绿体功能的光响应控制。已知大多数氧化还氧蛋白在光线下以依赖硫氧还蛋白（Trx）的方式被还原激活，但其调控网络仍不完全清楚。使用生化程序，我们在这里展示了一种特殊形式的磷酸果糖激酶（PFK）是一种新的氧化还原调节蛋白，其活性在还原时被抑制。PFK是糖酵解途径中的关键酶。在拟南芥中，PFK5靶向叶绿体，在n端区域独特地包含一个含有两个Cys残基（Cys152和Cys157）的插入序列。使用巯基修饰试剂进行的氧化还原移位实验表明，PFK5可以被一种特定类型的Trx，即Trx-f有效地还原。PFK5酶活性随着trx -f依赖性的减少而降低。PFK5的氧化还原调控是双向的；PFK5也通过最近发现的Trx-like2/2-Cys过氧化物还蛋白途径被氧化和激活。基于质谱的肽图谱分析显示，Cys152和Cys157对PFK5分子内二硫键的形成至关重要。一项定点诱变研究进一步支持了Cys152和Cys157参与PFK5氧化还原调控。PFK5催化果糖1,6-双磷酸酶（FBPase）的逆反应，而FBPase被Trx-f特异性地还原和激活。我们的数据表明，PFK5氧化还原调控与FBPase一起构成了叶绿体中光/暗代谢开关的检查点。
Various proteins in plant chloroplasts are subject to thiol-based redox regulation, allowing light-responsive control of chloroplast functions. Most redox-regulated proteins are known to be reductively activated in the light in a thioredoxin (Trx)-dependent manner, but its regulatory network remains incompletely understood. Using a biochemical procedure, we here show that a specific form of phosphofructokinase (PFK) is a novel redox-regulated protein whose activity is suppressed upon the reduction. PFK is a key enzyme in the glycolytic pathway. In Arabidopsis thaliana, PFK5 is targeted to chloroplasts and uniquely contains an insertion sequence harboring two Cys residues (Cys152 and Cys157) in the N-terminal region. Redox shift assays using a thiol-modifying reagent indicated that PFK5 is efficiently reduced by a specific type of Trx, namely, Trx-f. PFK5 enzyme activity was lowered with the Trx-f-dependent reduction. PFK5 redox regulation was bidirectional; PFK5 was also oxidized and activated by the recently identified Trx-like2/2-Cys peroxiredoxin pathway. Mass spectrometry-based peptide mapping analysis revealed that Cys152 and Cys157 are critical for the intramolecular disulfide bond formation in PFK5. The involvement of Cys152 and Cys157 in PFK5 redox regulation was further supported by a site-directed mutagenesis study. PFK5 catalyzes the reverse reaction of fructose 1,6-bisphosphatase (FBPase), which is reduced and activated specifically by Trx-f. Our data suggest that PFK5 redox regulation, together with that of FBPase, constitutes a checkpoint for switching light/dark metabolism in chloroplasts.