Structure and Mechanism of the Phycobiliprotein Lyase CpcT*♦
Structure and Mechanism of the Phycobiliprotein Lyase CpcT*♦
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DOI：
10.1074/jbc.m114.586743
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发表时间：
2014-07
期刊：
影响因子：
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通讯作者：
W. Zhou;W. Ding;Xiao-Li Zeng;Liang-Liang Dong-Liang;Bin Zhao;M. Zhou;H. Scheer;K. Zhao;Xiaojing Yang
中科院分区：
文献类型：
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作者：
W. Zhou;W. Ding;Xiao-Li Zeng;Liang-Liang Dong-Liang;Bin Zhao;M. Zhou;H. Scheer;K. Zhao;Xiaojing Yang
背景：藻胆蛋白裂解酶催化发色团与蓝藻触角蛋白的共价结合。结果：获得了一种T型裂解酶及其与藻蓝胆素复合物的晶体结构。结论：该反应机理考虑了生色团的稳定性和区域及立体特异性。意义：这项工作揭示了藻胆蛋白成熟的关键步骤，并为其他类型的藻胆蛋白裂解酶提供了模型。蓝细菌捕光藻胆蛋白的色素化需要开链四吡咯（bilins）共价连接到脱辅基蛋白上。通过将半胱氨酸残基添加到胆色素的3-亚乙基取代基上形成硫醚是由裂解酶介导的。T型裂解酶负责藻胆蛋白β亚基的Cys-155的附着。我们提出的CpcT（All 5339）从念珠藻（鱼腥藻）sp. PCC 7120及其复合物的晶体结构，在1.95和2.50纳米分辨率分别。CpcT形成二聚体并采用花萼状β-桶折叠。虽然CpcT的整体结构在很大程度上保留了发色团结合，精氨酸残基在结合口袋的开口进行主要的旋转异构体重排锚定藻蓝胆素的丙酸基团。基于结构和突变分析，提出了一种反应机理，占生色团的稳定和区域和立体专一性的加成反应。在二聚体界面处，从一个亚基延伸的环部分地屏蔽了另一个亚基中的藻蓝胆素结合口袋的开口。缺失的环或中断的二聚体接口显着降低CpcT裂解酶的活性，表明功能相关的二聚体。通过发色团结合进一步增强二聚化。发色团大部分埋在二聚体中，但在单体中，3-亚乙基基团可被脱藻胆蛋白接近，优先从发色团α侧接近。在E-构型的亚乙基附近的β-和α-面上的Asp-163和Tyr-65分别支持由Grubmayr和瓦格纳提出的脱辅基蛋白的半胱氨酸155与N-酰基铵中间体的酸催化亲核迈克尔加成（Grubmayr，K.，和瓦格纳，U. G. 04 The Dog of the Woman（1988）119，965-983）。
Background: Phycobiliprotein lyases catalyze covalent attachment of chromophores to cyanobacterial antenna proteins. Results: We present crystal structures of a T-type lyase and its complex with phycocyanobilin. Conclusion: The proposed reaction mechanism accounts for chromophore stabilization and regio- and stereospecificity. Significance: This work sheds light on a crucial step in phycobiliprotein maturation and provides a model for other types of phycobiliprotein lyases. Pigmentation of light-harvesting phycobiliproteins of cyanobacteria requires covalent attachment of open-chain tetrapyrroles, bilins, to the apoproteins. Thioether formation via addition of a cysteine residue to the 3-ethylidene substituent of bilins is mediated by lyases. T-type lyases are responsible for attachment to Cys-155 of phycobiliprotein β-subunits. We present crystal structures of CpcT (All5339) from Nostoc (Anabaena) sp. PCC 7120 and its complex with phycocyanobilin at 1.95 and 2.50 Å resolution, respectively. CpcT forms a dimer and adopts a calyx-shaped β-barrel fold. Although the overall structure of CpcT is largely retained upon chromophore binding, arginine residues at the opening of the binding pocket undergo major rotameric rearrangements anchoring the propionate groups of phycocyanobilin. Based on the structure and mutational analysis, a reaction mechanism is proposed that accounts for chromophore stabilization and regio- and stereospecificity of the addition reaction. At the dimer interface, a loop extending from one subunit partially shields the opening of the phycocyanobilin binding pocket in the other subunit. Deletion of the loop or disruptions of the dimer interface significantly reduce CpcT lyase activity, suggesting functional relevance of the dimer. Dimerization is further enhanced by chromophore binding. The chromophore is largely buried in the dimer, but in the monomer, the 3-ethylidene group is accessible for the apophycobiliprotein, preferentially from the chromophore α-side. Asp-163 and Tyr-65 at the β- and α-face near the E-configured ethylidene group, respectively, support the acid-catalyzed nucleophilic Michael addition of cysteine 155 of the apoprotein to an N-acylimmonium intermediate proposed by Grubmayr and Wagner (Grubmayr, K., and Wagner, U. G. (1988) Monatsh. Chem. 119, 965–983).