MOLECULAR-CLONING, CHARACTERIZATION, AND FUNCTIONAL EXPRESSION OF RAT OXIDOSQUALENE CYCLASE CDNA
MOLECULAR-CLONING, CHARACTERIZATION, AND FUNCTIONAL EXPRESSION OF RAT OXIDOSQUALENE CYCLASE CDNA
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DOI：
10.1073/pnas.92.20.9274
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发表时间：
1995-09-26
影响因子：
11.1
通讯作者：
PRESTWICH, GD
中科院分区：
文献类型：
--
作者：
ABE, I;PRESTWICH, GD
编码大鼠氧化角鲨烯羊毛甾醇环化酶的cDNA；克隆(S) -2,3-环氧奎烯突变酶（环化，羊毛甾醇形成），EC 5.4.99.7]，采用PCR扩增、基于纯化酶内部氨基酸序列的引物、寡核苷酸杂交筛选cDNA文库的方法进行测序。2199 bp的开放阅读框编码M(r) 83321蛋白，包含733个氨基酸。推导出的大鼠酶的氨基酸序列与已知的酵母和植物氧化角鲨烯环化酶（OSCs）具有显著的同源性（39-44%），与两种细菌氧化角鲨烯环化酶（EC 5.4.99.-）仍保持17-26%的同源性。与其他环化酶一样，这种大鼠酶富含芳香氨基酸，并含有5个所谓的QW基序，即具有重复β -链旋转基序的高度保守区域。29-甲基-2,3-氧化角鲨烯（29-MOS）的结合位点序列在所有已知的osc中都是保守的，这是一种基于机制的脊椎动物环化酶特异性不可逆抑制剂。亲水性图显示了一个相当亲水的n端区域和疏水信号肽的缺失。出乎意料的是，这种微粒体膜相关酶没有明确划定的跨膜结构域。构建全长cDNA并将其亚克隆到pYEUra3质粒中，在大肠杆菌细胞中选择，用于转化osc缺陷型酿酒酵母SGL9菌株。表达的重组大鼠OSC被基于机制的抑制剂[H-3] 29-MOS高效标记。
cDNA encoding rat oxidosqualene lanosterol-cyclase [Lanosterol synthase; (S) -2,3-epoxysqualene mutase (cyclizing, lanosterol-forming), EC 5.4.99.7] was cloned and sequenced by a combination of PCR amplification, using primers based on internal amino acid sequence of the purified enzyme, and cDNA library screening by oligonucleotide hybridization. An open reading frame of 2199 bp encodes a M(r)83,321 protein with 733 amino acids. The deduced amino acid sequence of the rat enzyme showed significant homology to the known oxidosqualene cyclases (OSCs) from yeast and plant (39-44% identity) and still retained 17-26% identity to two bacterial squalene cyclases (EC 5.4.99.-). Like other cyclases, the rat enzyme is rich in aromatic amino acids and contains five so-called QW motifs, highly conserved regions with a repetitive beta-strand turn motif, The binding site sequence for the 29-methylidene-2,3-oxidosqualene (29-MOS), a mechanism-based irreversible inhibitor specific for the vertebrate cyclase, is well-conserved in all known OSCs. The hydropathy plot revealed a rather hydrophilic N-terminal region and the absence of a hydrophobic signal peptide. Unexpectedly, this microsomal membrane-associated enzyme showed no clearly delineated transmembrane domain. A full-length cDNA was constructed and subcloned into a pYEUra3 plasmid, selected in Escherichia coli cells, and used to transform the OSC-deficient uracil-auxotrophic SGL9 strain of Saccharomyces cerevisiae. The recombinant rat OSC expressed was efficiently labeled by the mechanism-based inhibitor [H-3] 29-MOS.