CAMS Key Laboratory of Synthetic Biology for Drug Innovation, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050, China.
NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050, China.
Appl Microbiol Biotechnol. 2024 May 8;108(1):326. doi: 10.1007/s00253-024-13161-y.
Aspartyl dipeptidase (dipeptidase E) can hydrolyze Asp-X dipeptides (where X is any amino acid), and the enzyme plays a key role in the degradation of peptides as nutrient sources. Dipeptidase E remains uncharacterized in Streptomyces. Orf2 from Streptomyces sp. 139 is located in the exopolysaccharide biosynthesis gene cluster, which may be a novel dipeptidase E with "S134-H170-D198" catalytic triad by sequence and structure comparison. Herein, recombinant Orf2 was expressed in E. coli and characterized dipeptidase E activity using the Asp-ρNA substrate. The optimal pH and temperature for Orf2 are 7.5 and 40 ℃; Vmax and Km of Orf2 are 0.0787 mM·min and 1.709 mM, respectively. Orf2 exhibits significant degradation activities to Asp-Gly-Gly, Asp-Leu, Asp-His, and isoAsp-Leu and minimal activities to Asp-Pro and Asp-Ala. Orf2 contains a Ser-His-Asp catalytic triad characterized by point mutation. In addition, the Asp147 residue of Orf2 is also proven to be critical for the enzyme's activity through molecular docking and point mutation. Transcriptome analysis reveals the upregulation of genes associated with ribosomes, amino acid biosynthesis, and aminoacyl-tRNA biosynthesis in the orf2 mutant strain. Compared with the orf2 mutant strain and WT, the yield of crude polysaccharide does not change significantly. However, crude polysaccharides from the orf2 mutant strain exhibit a wider range of molecular weight distribution. The results indicate that the Orf2 links nutrient stress to secondary metabolism as a novel dipeptidase E. KEY POINTS: • A novel dipeptidase E with a Ser-His-Asp catalytic triad was characterized from Streptomyces sp. 139. • Orf2 was involved in peptide metabolism both in vitro and in vivo. • Orf2 linked nutrient stress to mycelia formation and secondary metabolism in Streptomyces.
天冬氨酰二肽酶（二肽酶 E）可以水解 Asp-X 二肽（其中 X 是任何氨基酸），该酶在肽作为营养源的降解中起关键作用。链霉菌中尚未鉴定出二肽酶 E。链霉菌 139 中的 Orf2 位于胞外多糖生物合成基因簇中，通过序列和结构比较，它可能是一种新型的具有“S134-H170-D198”催化三联体的二肽酶 E。在此，通过 Asp-ρNA 底物表达重组 Orf2 并对其二肽酶 E 活性进行了表征。Orf2 的最适 pH 和温度分别为 7.5 和 40℃；Orf2 的 Vmax 和 Km 分别为 0.0787 mM·min 和 1.709 mM。Orf2 对 Asp-Gly-Gly、Asp-Leu、Asp-His 和 isoAsp-Leu 具有显著的降解活性，对 Asp-Pro 和 Asp-Ala 的活性最小。Orf2 含有一个由点突变表征的 Ser-His-Asp 催化三联体。此外，还通过分子对接和点突变证明 Orf2 的 Asp147 残基对酶的活性也很关键。转录组分析表明，orf2 突变株中与核糖体、氨基酸生物合成和氨酰-tRNA 生物合成相关的基因上调。与 orf2 突变株和 WT 相比，粗多糖的产量没有明显变化。然而，orf2 突变株的粗多糖的分子量分布范围更广。结果表明，Orf2 作为一种新型的二肽酶 E 将营养胁迫与次级代谢联系起来。 关键点： • 从链霉菌 139 中鉴定出具有 Ser-His-Asp 催化三联体的新型二肽酶 E。 • Orf2 参与了体外和体内的肽代谢。 • Orf2 将营养胁迫与链霉菌中的菌丝形成和次级代谢联系起来。
