Department of Biological Sciences, University of New Orleans, New Orleans, Louisiana, USA.
Department of Chemistry, Indiana University, Bloomington, Indiana, USA.
J Biol Chem. 2021 Jan-Jun;296:100031. doi: 10.1074/jbc.RA120.015289. Epub 2020 Nov 23.
Synechococcus cyanobacteria are widespread in the marine environment, as the extensive pigment diversity within their light-harvesting phycobilisomes enables them to utilize various wavelengths of light for photosynthesis. The phycobilisomes of Synechococcus sp. RS9916 contain two forms of the protein phycoerythrin (PEI and PEII), each binding two chromophores, green-light absorbing phycoerythrobilin and blue-light absorbing phycourobilin. These chromophores are ligated to specific cysteines via bilin lyases, and some of these enzymes, called lyase isomerases, attach phycoerythrobilin and simultaneously isomerize it to phycourobilin. MpeV is a putative lyase isomerase whose role in PEI and PEII biosynthesis is not clear. We examined MpeV in RS9916 using recombinant protein expression, absorbance spectroscopy, and tandem mass spectrometry. Our results show that MpeV is the lyase isomerase that covalently attaches a doubly linked phycourobilin to two cysteine residues (C50, C61) on the β-subunit of both PEI (CpeB) and PEII (MpeB). MpeV activity requires that CpeB or MpeB is first chromophorylated by the lyase CpeS (which adds phycoerythrobilin to C82). Its activity is further enhanced by CpeZ (a homolog of a chaperone-like protein first characterized in Fremyella diplosiphon). MpeV showed no detectable activity on the α-subunits of PEI or PEII. The mechanism by which MpeV links the A and D rings of phycourobilin to C50 and C61 of CpeB was also explored using site-directed mutants, revealing that linkage at the A ring to C50 is a critical step in chromophore attachment, isomerization, and stability. These data provide novel insights into β-PE biosynthesis and advance our understanding of the mechanisms guiding lyase isomerases.
聚球藻蓝细菌广泛存在于海洋环境中，其光捕获藻胆体中的广泛色素多样性使它们能够利用光合作用的各种波长的光。Synechococcus sp. RS9916 的藻胆体含有两种形式的藻红蛋白（PEI 和 PEII），每种蛋白结合两个发色团，即绿光吸收的藻红胆素和蓝光吸收的藻蓝胆素。这些发色团通过藻胆素裂合酶连接到特定的半胱氨酸上，其中一些酶称为裂合酶异构酶，它们将藻红胆素连接并同时将其异构化为藻蓝胆素。MpeV 是一种假定的裂合酶异构酶，但其在 PEI 和 PEII 生物合成中的作用尚不清楚。我们使用重组蛋白表达、吸收光谱和串联质谱法在 RS9916 中研究了 MpeV。我们的结果表明，MpeV 是将双连接的藻蓝胆素共价连接到 PEI（CpeB）和 PEII（MpeB）β-亚基的两个半胱氨酸残基（C50、C61）上的裂合酶异构酶。MpeV 活性需要 CpeB 或 MpeB 首先被裂合酶 CpeS （将藻红胆素添加到 C82）发色团化。CpeZ（首次在 Fremyella diplosiphon 中表征的伴侣样蛋白的同源物）进一步增强了其活性。MpeV 对 PEI 或 PEII 的α-亚基没有检测到活性。使用定点突变体还探索了 MpeV 将藻蓝胆素的 A 和 D 环连接到 CpeB 的 C50 和 C61 的机制，结果表明，C50 上 A 环的连接是发色团附着、异构化和稳定性的关键步骤。这些数据为β-PE 生物合成提供了新的见解，并加深了我们对指导裂合酶异构酶的机制的理解。
