Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, United States.
Biochemistry. 2013 Feb 5;52(5):889-901. doi: 10.1021/bi3016229. Epub 2013 Jan 23.
Biosynthesis of the dipeptide antibiotic bacilysin, encoded by the seven Bacillus subtilis genes bacA-G, involves diversion of flux from prephenate to the noncognate amino acid anticapsin. The anticapsin warhead is then ligated to the C-terminus of l-alanine to produce mature bacilysin. We have previously noted the formation of two diastereomers of tetrahydrotyrosine (4S- and 4R-H(4)Tyr) by tandem action of the four purified enzymes BacABGF. BacC (oxidase) and BacD (ligase) have been hypothesized to be remaining late stage enzymes in bacilysin biosynthesis. Using a combination of BacCD in vitro studies, B. subtilis deletion mutants, and isotopic feeding studies, we were able to determine that the H(4)Tyr diastereomers are actually shunt products that are not on-pathway to bacilysin biosynthesis. Dihydroanticapsin and dihydrobacilysin accumulate in extracts of a ΔbacC strain and are processed to anticapsin and then bacilysin upon addition of BacC and BacD, respectively. These results suggest the epoxide group in bacilysin is installed in an earlier step of bacilysin biosynthesis, while BacC oxidation of the C(7)-hydroxyl and the subsequent BacD ligation of anticapsin to l-Ala are the penultimate and ultimate steps of bacilysin biosynthesis, respectively.
枯草芽孢杆菌七基因 bacA-G 编码的二肽抗生素枯草菌素的生物合成涉及从预苯酸到非天然氨基酸反-衣康酸的通量分流。然后，反-衣康酸弹头被连接到 l-丙氨酸的 C 末端，以产生成熟的枯草菌素。我们之前已经注意到由四个纯化酶 BacABGF 的串联作用形成了四氢酪氨酸（4S-和 4R-H（4）Tyr）的两种非对映异构体。推测 BacC（氧化酶）和 BacD（连接酶）是枯草菌素生物合成的剩余晚期酶。通过 BacCD 的体外研究、枯草芽孢杆菌缺失突变体和同位素喂养研究的组合，我们能够确定 H（4）Tyr 非对映异构体实际上是分流产物，而不是枯草菌素生物合成的途径产物。二氢反-衣康酸和二氢枯草菌素在 ΔbacC 菌株的提取物中积累，并在添加 BacC 和 BacD 后分别被加工成反-衣康酸和枯草菌素。这些结果表明，枯草菌素中的环氧化物基团在枯草菌素生物合成的早期步骤中安装，而 BacC 对 C（7）-羟基的氧化和随后 BacD 将反-衣康酸连接到 l-丙氨酸上是枯草菌素生物合成的倒数第二步和最后一步，分别。
