Structure-Function Analysis of A Novel Oxidosqualene Cyclase Involved in the Biosynthesis Antibiotics

抗生素生物合成中涉及的新型氧化角鲨烯环化酶的结构-功能分析

• 批准号: 12680597
• 负责人: ABE Ikuro
• 金额: $ 2.37万
• 依托单位: Univ. Shizuoka
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680597/
• 关键词: Oxidosqualene Cyclase; Steroidal Antibiotie; Lanosterol; Protosterol; Enzyme; Strucuture Function Relationship; Enzyme Purification; Cloning; 変異酵素; ステロイド; オキシドスクアレン; トリテルペン; 生合成; ヘルボール酸

A cDNA for oxidosqualene cyclase (OSC) was cloned and sequenced from the fungus Cephalosporium caerulens, that produces a steroidal antibiotic, helvolic acid. A 2,280-bp open reading frame encoded a Mr 87,078 protein with 760 amino acids, whose sequence showed ca. 37% identity with those of OSCs fromrat and yeast. The cDNA was functionally expressed in the OSLC-deficient mutant GIL77 strain of Saccharomyces cerevisiae. The recombinant enzyme afforded lanosterol as a singlele product. A truncated recombinant enzyme (Δ49N) starting from the second methionine (M50) residue was completely inactive, suggesting that ca. 30 additional hydrophilic amino acid residues at the N-terminal are essential fox the folding of the enzyme. Furthermore,, the active site residues, H234 and D456 (numbering in S. cerevisiae OSLC), were chosen for site-directed mutagenesis experiments; H234E, H234Y, H234F, D456E, D456N, and D456H mutants were inactive, while H234W and H234K mutants retained lanosterol forming activity. Purification of native protosterol synthase from the fungi is now in progress.

氧化角鲨烯环化酶（OSC）的cDNA克隆和测序的真菌Cephalosporium caerulens，产生的甾体抗生素，helvolic酸。该基因的开放阅读框为2，280 bp，编码760个氨基酸，分子量为87，078。与大鼠和酵母OSCs的同源性为37%。该cDNA在酿酒酵母的OSLC缺陷突变体GIL 77菌株中功能性表达。该重组酶以单一产物的形式提供羊毛甾醇。从第二个甲硫氨酸（M50）残基开始的截短重组酶（Δ 49 N）完全无活性，表明ca.在N端增加了30个亲水性氨基酸残基，这对酶的折叠是必需的。此外，活性位点残基H234和D456（在S.选择酿酒酵母OSLC）进行定点诱变实验; H234 E、H234 Y、H234 F、D456 E、D456 N和D456 H突变体是无活性的，而H234 W和H234 K突变体保留羊毛甾醇形成活性。从真菌中纯化天然原甾醇合酶的工作正在进行中。

项目成果

Ikuro Abe: "Design and Synthesis of Active Site Probes for Squalene Cyclase Enzymes"J. Synth. Org. Chem. Jpn.. 58. 745-755 (2001)

阿部郁郎：“角鲨烯环化酶活性位点探针的设计与合成”J。

Ikuro Abe, Katsuki Naito, Yosiharu Takagi, Hiroshi Noguchi: "Molecular Cloning, Expression, and Site-Directed mutations of Oxidosqualene Cyclase from Cephalosporium caerulens"Biochem. Biophys. Acta.. 1522. 67-73 (2001)

Ikuro Abe、Katsuki Naito、Yosiharu Takagi、Hiroshi Noguchi：“来自蓝头孢菌的氧化角鲨烯环化酶的分子克隆、表达和定点突变”Biochem。

I.Abe: "Molecular Cloning Expression and Site-directed Mutagenesis of Oxdosqulene Cyalose from Cepharosporium caerulens"Biochem. Biophys Acta. 1522. 67-73 (2001)

I.Abe：“来自 Cepharosporium caerulens 的 Oxdosqulene Cyalose 的分子克隆表达和定点诱变”Biochem。

I.Abe: "Design and Synthesis of Active Site Probes for Squalene Cyclase Enzymes"J. Synth. Org. Chem. Jpn. 58. 745-755 (2000)

I.Abe：“角鲨烯环化酶活性位点探针的设计与合成”J。

Ikuro Abe: "Bioorganic Studies on Oxidosqualene Cyclases"Advances in Pharmaceutical Sciences. 18 (in press). (2002)

阿部郁郎：“氧化角鲨烯环化酶的生物有机研究”制药科学进展。