Exchange of a single amino acid residue in the cryptophyte phycobiliprotein lyase GtCPES expands its substrate specificity
Exchange of a single amino acid residue in the cryptophyte phycobiliprotein lyase GtCPES expands its substrate specificity
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DOI：
10.1101/2020.03.31.018853
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发表时间：
2020-03
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影响因子：
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通讯作者：
Natascha Tomazic;K. Overkamp;Marco Aras;A. Pierik;E. Hofmann;N. Frankenberg‐Dinkel
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文献类型：
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作者：
Natascha Tomazic;K. Overkamp;Marco Aras;A. Pierik;E. Hofmann;N. Frankenberg‐Dinkel
隐藻是少数几种利用藻胆蛋白(PBP)在氧合光合作用中捕光的真核生物之一。与蓝藻PBP组织成大的膜相关超复合体不同，来自隐芽植物的藻胆体可溶于叶绿体类囊体腔内。它们的捕光能力是由于几个开链四吡咯发色团(藻胆蛋白)的共价连接。Guillardia theta利用PBP藻红蛋白PE545和15，16-二氢胆绿素(DHBV)以及藻红蛋白(PEB)作为发色团。到目前为止，隐芽植物PBP的组装还不完全清楚，但涉及PBP裂解酶的作用，如蓝藻PBP所示。PBP裂解酶有助于生色团以正确的构型和立体化学方式附着。在这里，我们提出了真核生物S类型的PBP裂解酶GtCPES的功能特性。我们发现GtCPES介导的PEB转移和共价连接到海洋原氯球菌MED4的受体β亚单位(PmCpeB)保守的Cys82上。基于先前求解的晶体结构，利用定点突变技术对GtCPES结合口袋进行了研究。从而鉴定了参与藻胆素结合和转运的氨基酸残基。有趣的是，单一氨基酸残基Met67到Ala的交换可能通过扩大底物结合口袋而延长了对藻蓝胆素(PCB)的底物专一性。突变体GtCPES_M67A在体外和体内都能与PEB和PCB结合，形成稳定的、彩色的复合体，在大肠杆菌中产生。GtCPES_M67A能够介导多氯联苯转移到PmCpeB的Cys82。根据我们的数据，我们推测，单个氨基酸残基决定了藻红蛋白S型裂解酶的BLIN专一性，但额外的因素调节了向目标蛋白的移交。
Cryptophyte algae are among the few eukaryotes that employ phycobiliproteins (PBP) for light harvesting during oxygenic photosynthesis. In contrast to the cyanobacterial PBP that are organized in large membrane-associated super complexes, the phycobilisomes, those from cryptophytes are soluble within the chloroplast thylakoid lumen. Their light-harvesting capacity is due to covalent linkage of several open-chain tetrapyrrole chromophores (phycobilins). Guillardia theta utilizes the PBP phycoerythrin PE545 with 15,16-dihydrobiliverdin (DHBV) in addition to phycoerythrobilin (PEB) as chromophores. Thus far, the assembly of cryptophyte PBPs is not yet completely understood but involves the action of PBP-lyases as shown for cyanobacterial PBP. PBP-lyases facilitate the attachment of the chromophore in the right configuration and stereochemistry. Here we present the functional characterization of eukaryotic S-type PBP lyase GtCPES from G. theta. We show GtCPES mediated transfer and covalent attachment of PEB to the conserved Cys82 of the acceptor PBP β-subunit (PmCpeB) of Prochlorococcus marinus MED4. Based on the previously solved crystal structure, the GtCPES binding pocket was investigated using site-directed mutagenesis. Thereby, amino acid residues involved in phycobilin binding and transfer were identified. Interestingly, exchange of a single amino acid residue Met67 to Ala extended the substrate specificity to phycocyanobilin (PCB) likely by enlarging the substrate-binding pocket. Variant GtCPES_M67A binds both PEB and PCB forming a stable, colorful complex in vitro and in vivo produced in Escherichia coli. GtCPES_M67A is able to mediate PCB transfer to Cys82 of PmCpeB. Based on our data we postulate that a single amino acid residue determines the bilin-specificity of phycoerythrin S-type lyases but that additional factors regulate hand over to the target protein.