Cryptic Production of trans-3-Hydroxyproline in Echinocandin B Biosynthesis
Cryptic Production of trans-3-Hydroxyproline in Echinocandin B Biosynthesis
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DOI：
10.1128/aem.02370-17
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发表时间：
2018-01
影响因子：
4.4
通讯作者：
Johanna Mattay;S. Houwaart;Wolfgang Hüttel
中科院分区：
文献类型：
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作者：
Johanna Mattay;S. Houwaart;Wolfgang Hüttel
棘白菌素是由真菌产生的抗真菌非核糖体六肽。其中两种氨基酸是羟基-1-脯氨酸：反式-4-羟基-1-脯氨酸，在大多数棘白菌素结构中，是（反式-2，3）-3-羟基-（反式-2，4）-4-甲基-1-脯氨酸。在由Glarea lozoyensis生物合成棘白菌素的情况下，两种氨基酸都存在于肺炎白菌素A0中，而在肺炎白菌素B 0中，后者残基被反式-3-羟基-1-脯氨酸（3-Hyp）取代。我们最近报道，这三种氨基酸都是由2-酮戊二酸依赖性脯氨酸羟化酶GloF产生的。在曲霉属物种的棘白菌素B生物合成中，3-Hyp衍生物尚未报道。在这里，我们描述了异源生产和动力学特性的HtyE，2-酮戊二酸依赖性脯氨酸羟化酶从棘白菌素B生物合成簇在粗冠曲霉。令人惊讶的是，用HtyE进行的l-脯氨酸羟基化导致比用GloF更高比例（约30%）的3-Hyp。这表明棘白菌素B生物合成中甲基化3-羟脯氨酸的选择性仅仅是由于非核糖体肽合成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-1-脯氨酸在与HtyE长时间孵育后在甲基处缓慢地进一步氧化，得到3-羟基-4-羟甲基-1-脯氨酸。据报道，这种二羟基化的氨基酸是隐孢子虫产生的棘白菌素隐白菌素的组成部分。细菌和真菌的次级代谢产物通常由不同基因簇编码的生物合成酶产生。通常，每种酶催化一个生物合成步骤，但也可能有多个反应。Pneumocandins A0和B 0由Glarea lozoyensis真菌产生。它们属于棘白菌素家族，一组非核糖体环状脂肽，具有很强的抗真菌活性。化学衍生物是治疗系统性真菌感染的重要药物。我们最近已经表明，在pneumocandins A0和B 0的生物合成中，三个羟脯氨酸结构单元由一个脯氨酸羟化酶提供。在这里，我们证明，脯氨酸羟化酶从棘白菌素B生物合成的粗冠曲霉产生相同的羟脯氨酸，反式-3-羟脯氨酸的比例增加。然而，棘白菌素B的生物合成不需要反式-3-羟脯氨酸;它的形成仍然是神秘的。虽然人们只能推测这一意外发现的进化背景，但G。lozoyensis和A. Pachycristatus提供了对肽抗生素生物合成的不寻常的洞察，即羟化酶的选择性和非核糖体肽合成酶的底物特异性之间的复杂相互作用。
ABSTRACT Echinocandins are antifungal nonribosomal hexapeptides produced by fungi. Two of the amino acids are hydroxy-l-prolines: trans-4-hydroxy-l-proline and, in most echinocandin structures, (trans-2,3)-3-hydroxy-(trans-2,4)-4-methyl-l-proline. In the case of echinocandin biosynthesis by Glarea lozoyensis, both amino acids are found in pneumocandin A0, while in pneumocandin B0 the latter residue is replaced by trans-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 Aspergillus 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 Aspergillus pachycristatus. 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 Cryptosporiopsis. IMPORTANCE 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 A0 and B0 are produced by the fungus Glarea lozoyensis. 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 A0 and B0, three hydroxyproline building blocks are provided by one proline hydroxylase. Here we demonstrate that the proline hydroxylase from echinocandin B biosynthesis in Aspergillus pachycristatus produces the same hydroxyprolines, with an increased proportion of trans-3-hydroxyproline. However, echinocandin B biosynthesis does not require trans-3-hydroxyproline; its formation remains cryptic. While one can only speculate on the evolutionary background of this unexpected finding, proline hydroxylation in G. lozoyensis and A. pachycristatus 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.