聚基二重键的生物合成. 皮克罗米/甲基多基化合成酶含脱水酶的模块2的机械分析
Jiaquan Wu1, Toby J Zaleski, Chiara Valenzano
1Department of Chemistry, Box H, Brown University, Providence, Rhode Island 02912-9108, USA.
Journal of the American Chemical Society
|December 8, 2005
概括
这项研究阐明了皮克罗米/甲基合成酶 (PICS) 模块2中的多基基链延长周期. 研究人员确定了关键中间体的结构,揭示了脱水酶和缩酶域在宏类抗生素生物合成中的作用.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 有机化学 有机化学
背景情况:
- 皮克罗米/甲基合成酶 (PICS) 是一种模块化聚基化合成酶 (PKS),对于宏类抗生素前体生物合成至关重要.
- 含有脱水酶 (DH) 域的 PICS 模块2 使用马洛尼尔-CoA 形成不和三基甲酸中间体.
- 了解PKS的催化机制对于设计新型生物活性化合物至关重要.
研究的目的:
- 描述复合PICS模块2+TE的催化活性和基质特异性.
- 确定由PICS模块2形成的多基基介质的立体化学和结构.
- 阐明脱水酶 (DH) 和缩酶 (KR) 域在PKS催化循环中的作用.
主要方法:
- 在大肠杆菌中表达和净化复合PICS模块2+TE.
- 使用马洛尼尔-CoA和特定的二甲基底 (SNAC衍生物) 的酶分析.
- 使用质谱和NMR分析反应产品以确定结构和立体化学.
- 位点定向的突变发生,使DH和KR域失活,用于功能分析.
主要成果:
- PICS模块2+TE产生了三酸和三酸乳的混合物,乳通过竞争的硫酶催化循环形成.
- 该酶表现出对合成二甲基-SNAC基质的强烈偏好,而不是对抗异构体.
- DH域的非激活仅产生了三基酸乳,而KR的非激活产生了未减少的酸乳.
结论:
- 本研究确定了 DH 含有 PKS 模块延长周期中中间体的结构和立体化学.
- 这些发现证实了活性部位残留在KR和DH领域对立体化学控制的关键作用.
- 结果提供了对多基基生物合成途径和合成生物学应用潜力的基本见解.
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