Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
Proc Natl Acad Sci U S A. 2010 Aug 31;107(35):15391-6. doi: 10.1073/pnas.1007953107. Epub 2010 Aug 16.
The biosynthesis of chloramphenicol requires a beta-hydroxylation tailoring reaction of the precursor L-p-aminophenylalanine (L-PAPA). Here, it is shown that this reaction is catalyzed by the enzyme CmlA from an operon containing the genes for biosynthesis of L-PAPA and the nonribosomal peptide synthetase CmlP. EPR, Mössbauer, and optical spectroscopies reveal that CmlA contains an oxo-bridged dinuclear iron cluster, a metal center not previously associated with nonribosomal peptide synthetase chemistry. Single-turnover kinetic studies indicate that CmlA is functional in the diferrous state and that its substrate is L-PAPA covalently bound to CmlP. Analytical studies show that the product is hydroxylated L-PAPA and that O(2) is the oxygen source, demonstrating a monooxygenase reaction. The gene sequence of CmlA shows that it utilizes a lactamase fold, suggesting that the diiron cluster is in a protein environment not previously known to effect monooxygenase reactions. Notably, CmlA homologs are widely distributed in natural product biosynthetic pathways, including a variety of pharmaceutically important beta-hydroxylated antibiotics and cytostatics.
氯霉素的生物合成需要前体 L-对氨基苯丙氨酸 (L-PAPA) 的β-羟化修饰反应。本文表明，该反应由包含 L-PAPA 生物合成基因和非核糖体肽合成酶 CmlP 的操纵子中的酶 CmlA 催化。EPR、穆斯堡尔和光学光谱学表明，CmlA 含有一个氧桥双核铁簇，这是一个以前与非核糖体肽合成酶化学无关的金属中心。单轮动力学研究表明，CmlA 在二价铁状态下具有功能，其底物是与 CmlP 共价结合的 L-PAPA。分析研究表明产物是羟化的 L-PAPA，并且 O(2) 是氧源，证明了单加氧酶反应。CmlA 的基因序列表明它利用了一个内酰胺酶折叠，这表明双核铁簇处于一种以前未知的蛋白质环境中，该环境会影响单加氧酶反应。值得注意的是，CmlA 同源物广泛存在于天然产物生物合成途径中，包括各种具有重要药用价值的β-羟化抗生素和细胞抑制剂。
