Institute of Pharmaceutical Sciences, Department of Pharmaceutical and Medicinal Chemistry, University of Freiburg, Freiburg, Germany.
Institute of Pharmaceutical Sciences, Department of Pharmaceutical and Medicinal Chemistry, University of Freiburg, Freiburg, Germany
Appl Environ Microbiol. 2018 Mar 19;84(7). doi: 10.1128/AEM.02370-17. Print 2018 Apr 1.
Echinocandins are antifungal nonribosomal hexapeptides produced by fungi. Two of the amino acids are hydroxy-l-prolines: -4-hydroxy-l-proline and, in most echinocandin structures, (-2,3)-3-hydroxy-(-2,4)-4-methyl-l-proline. In the case of echinocandin biosynthesis by , both amino acids are found in pneumocandin A, while in pneumocandin B the latter residue is replaced by -3-hydroxy-l-proline (3-Hyp). We have recently reported that all three amino acids are generated by the 2-oxoglutarate-dependent proline hydroxylase GloF. In echinocandin B biosynthesis by species, 3-Hyp derivatives have not been reported. Here we describe the heterologous production and kinetic characterization of HtyE, the 2-oxoglutarate-dependent proline hydroxylase from the echinocandin B biosynthetic cluster in Surprisingly, l-proline hydroxylation with HtyE resulted in an even higher proportion (∼30%) of 3-Hyp than that with GloF. This suggests that the selectivity for methylated 3-Hyp in echinocandin B biosynthesis is due solely to a substrate-specific adenylation domain of the nonribosomal peptide synthetase. Moreover, we observed that one product of HtyE catalysis, 3-hydroxy-4-methyl-l-proline, is slowly further oxidized at the methyl group, giving 3-hydroxy-4-hydroxymethyl-l-proline, upon prolonged incubation with HtyE. This dihydroxylated amino acid has been reported as a building block of cryptocandin, an echinocandin produced by Secondary metabolites from bacteria and fungi are often produced by sets of biosynthetic enzymes encoded in distinct gene clusters. Usually, each enzyme catalyzes one biosynthetic step, but multiple reactions are also possible. Pneumocandins A and B are produced by the fungus They belong to the echinocandin family, a group of nonribosomal cyclic lipopeptides that exhibit a strong antifungal activity. Chemical derivatives are important drugs for the treatment of systemic fungal infections. We have recently shown that in the biosynthesis of pneumocandins A and B, three hydroxyproline building blocks are provided by one proline hydroxylase. Here we demonstrate that the proline hydroxylase from echinocandin B biosynthesis in produces the same hydroxyprolines, with an increased proportion of -3-hydroxyproline. However, echinocandin B biosynthesis does not require -3-hydroxyproline; its formation remains cryptic. While one can only speculate on the evolutionary background of this unexpected finding, proline hydroxylation in and provides an unusual insight into peptide antibiotic biosynthesis-namely, the complex interplay between the selectivity of a hydroxylase and the substrate specificity of a nonribosomal peptide synthetase.
棘白菌素是真菌产生的抗真菌非核糖体六肽。两种氨基酸是羟脯氨酸：-4-羟脯氨酸和，在大多数棘白菌素结构中，（-2,3）-3-羟基-（-2,4）-4-甲基-l-脯氨酸。在通过产生棘白菌素的生物合成中，这两种氨基酸都存在于肺囊菌素 A 中，而在肺囊菌素 B 中，后一种残基被-3-羟基-l-脯氨酸（3-Hyp）取代。我们最近报道，所有三种氨基酸都是由 2-氧戊二酸依赖性脯氨酸羟化酶 GloF 生成的。在通过 产生的棘白菌素 B 生物合成中，尚未报道 3-Hyp 衍生物。在这里，我们描述了来自棘白菌素 B 生物合成簇的 2-氧戊二酸依赖性脯氨酸羟化酶 HtyE 的异源生产和动力学特征。令人惊讶的是，与 GloF 相比，HtyE 对 l-脯氨酸的羟化导致 3-Hyp 的比例甚至更高（约 30%）。这表明棘白菌素 B 生物合成中对甲基化 3-Hyp 的选择性仅归因于非核糖体肽合酶的底物特异性腺苷酸化结构域。此外，我们观察到 HtyE 催化的一种产物，3-羟基-4-甲基-l-脯氨酸，在与 HtyE 长时间孵育后，在甲基上缓慢进一步氧化，生成 3-羟基-4-羟甲基-l-脯氨酸。这种二羟基氨基酸已被报道为 cryptocandin 的构建块，cryptocandin 是一种由 产生的棘白菌素。细菌和真菌的次生代谢产物通常由一组在不同基因簇中编码的生物合成酶产生。通常，每种酶都催化一个生物合成步骤，但也可能有多个反应。肺囊菌素 A 和 B 是由真菌 产生的。它们属于棘白菌素家族，是一组具有强抗真菌活性的非核糖体环状脂肽。化学衍生物是治疗系统性真菌感染的重要药物。我们最近表明，在肺囊菌素 A 和 B 的生物合成中，一种脯氨酸羟化酶提供了三个羟脯氨酸构建块。在这里，我们证明了棘白菌素 B 生物合成中的脯氨酸羟化酶在 中产生相同的羟脯氨酸，-3-羟基脯氨酸的比例增加。然而，棘白菌素 B 的生物合成并不需要 -3-羟基脯氨酸；它的形成仍然是隐藏的。虽然人们只能推测这一意外发现的进化背景，但 中的脯氨酸羟化作用和 中的非核糖体肽合酶的底物特异性提供了对抗生素生物合成的肽的复杂相互作用的独特见解。
