Crystal structure of type-III geranylgeranyl pyrophosphate synthase from Saccharomyces cerevisiae and the mechanism of product chain length determination
Crystal structure of type-III geranylgeranyl pyrophosphate synthase from Saccharomyces cerevisiae and the mechanism of product chain length determination
复制标题
DOI：
10.1074/jbc.m512886200
复制
发表时间：
2006-05-26
影响因子：
4.8
通讯作者：
Liang, Po-Huang
中科院分区：
文献类型：
--
作者：
Chang, Tao-Hsin;Guo, Rey-Ting;Liang, Po-Huang
牛儿基牛儿基焦磷酸合酶（GGPPs）催化法尼基焦磷酸与异戊烯基焦磷酸的缩合反应以产生C-20牛儿基牛儿基焦磷酸，其是类胡萝卜素、叶绿素、牛儿基牛儿基化蛋白质和古细菌醚连接脂质的前体。对于合成C-10-C-25产物的短链反式异戊二烯基转移酶，大体积氨基酸残基通常占据第一个DDXXD基序上游的第四或第五位置，以阻断最终产物的进一步延伸。然而，真核生物中的短链III型GDPP在这些位置缺乏任何大的氨基酸。在这项研究中，来自酿酒酵母的III型GGPPs的第一结构已被确定为1.98（A）以上的圆分辨率。该结构完全由15个通过连接环连接的α-螺旋组成，并围绕一个大的中心空腔排列α-螺旋。与反式异戊二烯基转移酶的其他已知结构不同，该GDPP的N-末端17个氨基酸（9-氨基酸螺旋A和随后的环）从螺旋核心突出到其他亚基中，并有助于紧密二聚体的形成。前9或17个氨基酸的缺失导致二聚体解离成单体，并且Delta（1 - 17）突变体显示酶活性丧失。在每个亚基中，由D、F、G、H和I α-螺旋包围的细长疏水缝隙在顶部包含两个DDXXD基序，用于与一个由Asp（75）、Asp（79）和四个水分子配位的Mg 2+结合。它在底部用Tyr（107）、Phe（108）和His（139）的三个大残基密封。与突变体H139A合成的主要产物C-30相比，突变体Y107A和F108A合成的产物主要是C-40和C-30，表明Tyr（107）在决定产物链长度方面起着最重要的作用。
Geranylgeranyl pyrophosphate synthase ( GGPPs) catalyzes a condensation reaction of farnesyl pyrophosphate with isopentenyl pyrophosphate to generate C-20 geranylgeranyl pyrophosphate, which is a precursor for carotenoids, chlorophylls, geranylgeranylated proteins, and archaeal ether-linked lipid. For short-chain trans-prenyltransferases that synthesize C-10-C-25 products, bulky amino acid residues generally occupy the fourth or fifth position upstream from the first DDXXD motif to block further elongation of the final products. However, the short-chain type-III GGPPs in eukaryotes lack any large amino acid at these positions. In this study, the first structure of type-III GGPPs from Saccharomyces cerevisiae has been determined to 1.98 (A) over circle resolution. The structure is composed entirely of 15 alpha-helices joined by connecting loops and is arranged with alpha-helices around a large central cavity. Distinct from other known structures of trans-prenyltransferases, the N-terminal 17 amino acids ( 9-amino acid helix A and the following loop) of this GGPPs protrude from the helix core into the other subunit and contribute to the tight dimer formation. Deletion of the first 9 or 17 amino acids caused the dissociation of dimer into monomer, and the Delta( 1 - 17) mutant showed abolished enzyme activity. In each subunit, an elongated hydrophobic crevice surrounded by D, F, G, H, and I alpha-helices contains two DDXXD motifs at the top for substrate binding with one Mg2+ coordinated by Asp(75), Asp(79), and four water molecules. It is sealed at the bottom with three large residues of Tyr(107), Phe(108), and His(139). Compared with the major product C-30 synthesized by mutant H139A, the products generated by mutant Y107A and F108A are predominantly C-40 and C-30, respectively, suggesting the most important role of Tyr(107) in determining the product chain length.