A Highly Conserved Basidiomycete Peptide Synthetase Produces a Trimeric Hydroxamate Siderophore
A Highly Conserved Basidiomycete Peptide Synthetase Produces a Trimeric Hydroxamate Siderophore
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DOI：
10.1128/aem.01478-17
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发表时间：
2017-11-01
影响因子：
4.4
通讯作者：
Hoffmeister, Dirk
中科院分区：
文献类型：
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作者：
Brandenburger, Eileen;Gressler, Markus;Hoffmeister, Dirk
模式白腐菌Ceriporiopsis（Gelatoporia）subver-mispora B编码推定的天然产物生物合成基因。其中包括七域非核糖体肽合成酶CsNPS 2的基因。它是迄今尚未表征的真菌VI型铁载体合成酶家族的成员，该家族高度保守且广泛分布于担子菌中。这些酶仅包括一个腺苷酸化（A）结构域，即，一个完整的肽合成酶模块和两个巯基化/缩合（T-C）双结构域部分模块，它们一起构成AT（1）C（1）T（2）C（2）T（3）C（3）结构域设置。全长CsNPS 2酶（274.5 kDa）异源产生的多聚组氨酸融合在尼日尔曲霉作为一种可溶性和活性蛋白。基于体外底物依赖性[P-32] ATP-焦磷酸放射性同位素交换测定的结果，N-5-乙酰基-N-5-羟基-L-鸟氨酸（L-AHO）和N-5-顺式-脱水甲羟戊酸基-N-5-羟基-L-鸟氨酸（L-AMHO）被接受为底物。全长holo-CsNPS 2催化三个L-AHO分子之间形成酰胺键，在体外释放线性L-AHO三聚体，称为担子铁蛋白，这是通过液相色谱-高分辨电喷雾电离-质谱分析验证。系统发育分析表明，VI型家庭铁载体合成酶是广泛存在于蘑菇和进化在一个共同的祖先的basidiomycetes.IMPORTANCE的basidiomycete非核糖体肽合成酶CsNPS 2代表一个成员的广泛分布，但以前未调查类（VI型）的真菌铁载体合成酶。与CsNPS 2直向同源的基因在担子菌的各个系统发育分支中高度保守。因此，我们的工作作为一个广泛适用的模式，铁载体生物合成和铁代谢在高等真菌。此外，我们的研究结果CsNPS 2的氨基酸底物偏好支持进一步了解真菌腺苷酸化结构域的底物选择性。在方法学上，该报告强调了基于尼日尔/SMXpress的系统作为以可溶性和活性形式异源表达> 250-kDa范围内的多模块担子菌生物合成酶的合适平台。
The model white-rot basidiomycete, Ceriporiopsis (Gelatoporia) subver-mispora B, encodes putative natural product biosynthesis genes. Among them is the gene for the seven-domain nonribosomal peptide synthetase CsNPS2. It is a member of the as-yet-uncharacterized fungal type VI siderophore synthetase family, which is highly conserved and widely distributed among the basidiomycetes. These enzymes include only one adenylation (A) domain, i.e., one complete peptide synthetase module, and two thiolation/condensation (T-C) didomain partial modules which together constitute an AT(1)C(1)T(2)C(2)T(3)C(3) domain setup. The full-length CsNPS2 enzyme (274.5 kDa) was heterologously produced as a polyhistidine fusion in Aspergillus niger as a soluble and active protein. N-5-acetyl-N-5-hydroxy-L-ornithine (L-AHO) and N-5-cis-anhydromevalonyl-N-5-hydroxy-L-ornithine (L-AMHO) were accepted as the substrates, based on results of an in vitro substrate-dependent [P-32] ATP-pyrophosphate radioisotope exchange assay. Full-length holo-CsNPS2 catalyzed amide bond formation between three L-AHO molecules to release the linear L-AHO trimer, called basidioferrin, as the product in vitro, which was verified by liquid chromatography-high-resolution electrospray ionization-mass spectrometry analysis. Phylogenetic analyses suggested that type VI family siderophore synthetases are widespread in mushrooms and evolved in a common ancestor of basidiomycetes.IMPORTANCE The basidiomycete nonribosomal peptide synthetase CsNPS2 represents a member of a widely distributed but previously uninvestigated class (type VI) of fungal siderophore synthetases. Genes orthologous to CsNPS2 are highly conserved across various phylogenetic clades of the basidiomycetes. Hence, our work serves as a broadly applicable model for siderophore biosynthesis and iron metabolism in higher fungi. Also, our results on the amino acid substrate preference of CsNPS2 support a further understanding of the substrate selectivity of fungal adenylation domains. Methodologically, this report highlights the Aspergillus niger/SMXpress- based system as a suitable platform to heterologously express multimodular basidiomycete biosynthesis enzymes in the > 250-kDa range in soluble and active form.