Jung L J, Chan C F, Glazer A N
Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
J Biol Chem. 1995 May 26;270(21):12877-84. doi: 10.1074/jbc.270.21.12877.
The rod substructures of the Anabaena sp. PCC 7120 phycobilisome contain the light harvesting proteins C-phycocyanin and phycoerythrocyanin (PEC). Even at low light intensities, PEC represents no more than 5% of the phycobilisome protein. The beta subunits of both proteins carry thioether-linked phycocyanobilin (PCB) at beta-Cys-82 and beta-Cys-155; however, C-phycocyanin has PCB at alpha-Cys-84 whereas PEC alpha subunit carries phycobiliviolin at this position. The Anabaena sp. PCC 7120 pec operon is made up of five genes. PecB and pecA encode the beta and alpha subunits of PEC, pecC encodes a linker polypeptide associated with PEC in the rod substructure, and pecE and pecF are genes of unknown function that show a high degree of homology to cpcE and cpcF, that encode a C-phycocyanin alpha subunit PCB lyase (Fairchild, C. D., Zhao, J., Zhou, J., Colson, S. E., Bryant, D. A., and Glazer, A. N. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7017-7021). Insertional mutants in pecE and pecF, and an interposon mutant in which a portion of both pecE and pecF was deleted, were constructed. All three types of mutants grew 1.3 times slower than wild-type under limiting light conditions and showed a 20% reduction in the PCB content of whole cells relative to chlorophyll alpha. Holo-PEC was missing from the phycobilisomes of all three types of mutants and the level of the PEC linker polypeptide was reduced relative to the wild-type. However, approximately 30% of the wild-type level of the PEC beta subunit was present in all of these phycobilisomes. In contrast, the PEC alpha subunit was barely detectable in the pecE and pecF mutants, but was present in the pecEF deletion mutant as a PCB-adduct in a 1:1 ratio with the PEC beta subunit. The identity of this "unnatural" adduct was confirmed by isolation of the subunit and amino-terminal sequencing. These biochemical results support the inference that pecE and pecF encode a PEC alpha subunit phycobiliviolin lyase, and, in conjunction with earlier findings, demonstrate that phycobiliprotein bilin lyases show high selectivity (rather than absolute specificity) for both the bilin and the polypeptide substrate.
鱼腥藻PCC 7120藻胆体的杆状亚结构包含光捕获蛋白C-藻蓝蛋白和藻红胆素蛋白（PEC）。即使在低光照强度下，PEC也仅占藻胆体蛋白的5% 。这两种蛋白的β亚基在β-Cys-82和β-Cys-155处携带硫醚连接的藻蓝胆素（PCB）；然而，C-藻蓝蛋白在α-Cys-84处有PCB，而PEC的α亚基在此位置携带藻胆紫素。鱼腥藻PCC 7120的pec操纵子由五个基因组成。PecB和pecA编码PEC的β和α亚基，pecC编码与杆状亚结构中的PEC相关的连接多肽，pecE和pecF是功能未知的基因，与编码C-藻蓝蛋白α亚基PCB裂合酶的cpcE和cpcF具有高度同源性（Fairchild, C. D., Zhao, J., Zhou, J., Colson, S. E., Bryant, D. A., and Glazer, A. N. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7017 - 7021）。构建了pecE和pecF的插入突变体以及一个同时缺失pecE和pecF部分序列的插入子突变体。在限制光照条件下，所有这三种类型的突变体生长速度比野生型慢1.3倍，并且相对于叶绿素α，全细胞中的PCB含量降低了20% 。所有这三种类型的突变体的藻胆体中均缺失全藻胆素蛋白PEC，并且PEC连接多肽的水平相对于野生型有所降低。然而，在所有这些藻胆体中都存在约30%野生型水平的PECβ亚基。相比之下，在pecE和pecF突变体中几乎检测不到PECα亚基，但在pecEF缺失突变体中，它以与PECβ亚基1:1的比例作为PCB加合物存在。通过亚基分离和氨基末端测序证实了这种“非天然”加合物的身份。这些生化结果支持了pecE和pecF编码PECα亚基藻胆紫素裂合酶的推断，并且与早期发现一起，证明了藻胆蛋白胆素裂合酶对胆素和多肽底物都具有高选择性（而非绝对特异性）。
