Department of Chemistry/Biochemistry, Philipps-University, Hans-Meerwein-Strasse, D-35032 Marburg, Germany.
J Am Chem Soc. 2009 Sep 23;131(37):13523-30. doi: 10.1021/ja9054417.
The antifungal and antimicrobial kutznerides, hexadepsipeptides composed of one alpha-hydroxy acid and five nonproteinogenic amino acids, are remarkable examples of the structural diversity found in nonribosomally produced natural products. They contain D-3-hydroxyglutamic acid, which is found in the threo and erythro isomers in mature kutznerides. In this study, two putative nonheme iron oxygenase enzymes, KtzO and KtzP, were recombinantly expressed, characterized biochemically in vitro, and found to stereospecifically hydroxylate the beta-position of glutamic acid. KtzO generates threo-L-hydroxyglutamic acid and KtzP catalyzes the formation of the erythro-isomer bound to the peptidyl carrier protein of the third module of the nonribosomal peptide synthetase KtzH. This module has a truncated adenylation domain and is unable to activate and incorporate glutamic acid. The lack of a functional adenylation domain in the third KtzH module is compensated in trans by the stand-alone adenylation domain KtzN, which activates and transfers glutamic acid onto the carrier of KtzH in the presence of the truncated adenylation domain and either KtzO or KtzP. A method that employs nonhydrolyzable coenzyme A analogs was developed and used to determine the kinetic parameters for KtzO- and KtzP-catalyzed hydroxylation of glutamic acid bound to the carrier protein. A detailed mechanism for the in trans compensation of the truncated adenylation domain and the stereospecific hydroxyglutamic acid generation and incorporation is presented. These insights may guide the use of KtzO/KtzP and KtzN or other in trans modification/restoration tools in biocombinatorial engineering approaches.
抗真菌和抗菌的 kutznerides 是由一个α-羟基酸和五个非蛋白质氨基酸组成的十六肽，是在非核糖体产生的天然产物中发现的结构多样性的显著例子。它们含有 D-3-羟基谷氨酸，在成熟的 kutznerides 中发现有苏式和赤式异构体。在这项研究中，两个假定的非血红素铁加氧酶酶 KtzO 和 KtzP 被重组表达，体外进行了生化特性表征，并发现它们能立体特异性地羟化谷氨酸的β位。KtzO 生成苏式-L-羟基谷氨酸，而 KtzP 催化与非核糖体肽合成酶 KtzH 的第三个模块的肽酰载体蛋白结合的赤式异构体的形成。该模块具有截短的腺苷酸化结构域，无法激活并掺入谷氨酸。第三个 KtzH 模块中缺乏功能性腺苷酸化结构域通过独立的腺苷酸化结构域 KtzN 进行反式补偿，该结构域在截短的腺苷酸化结构域和 KtzO 或 KtzP 的存在下激活并将谷氨酸转移到 KtzH 的载体上。开发了一种使用不可水解辅酶 A 类似物的方法，并用于确定 KtzO 和 KtzP 催化与载体蛋白结合的谷氨酸羟化的动力学参数。提出了一个详细的反式补偿截短的腺苷酸化结构域和立体特异性羟谷氨酸生成和掺入的机制。这些见解可能指导在生物组合工程方法中使用 KtzO/KtzP 和 KtzN 或其他反式修饰/恢复工具。
