土麴黴黑色素 Aspulvinone E 生合成之類非核醣體胜肽合成酶中 A domain 結構與功能探討

Structural and functional elucidation of adenylation domain of nonribosomal peptide synthetase-like protein in Aspulvinone E biosynthesis

李政祐(Cheng-Yu Li)

指導教授 ： 朱忠瀚

共同指導教授 ： 徐駿森(Chun-Hua Hsu)

國立臺灣大學/理學院/化學研究所/碩士(2022年)

https://doi.org/10.6342/NTU202202724

摘要

非核糖體肽合成酶 (nonribosomal peptide synthetase, NRPS) 為一類具有多個模組的大型蛋白質， 主要負責於製造出具有胜肽鍵結構的次級代謝物。而有一類型含有醌 (quinone) 結構的天然物，是經由另一種類非核糖體肽合成酶 (NRPS-like) 蛋白所製造。NRPS-like 蛋白是由 A-T-TE 三個 domain 所組成，且在 condensation 的階段時不具有 C domain 的參與。在 Aspergillus terreus 中，有一個黑色素 Aspulvinone E 被證實是由 NRPS-like 蛋白 ApvA 所製造。其中 L-tyrosine經轉胺酶 (aminotransferase) 而得到之產物 4-羥苯丙酮酸 (4-hydroxyphenylpyruvic acid, 4-HPPA) 將由 ApvA 中的 A domain (ApvA-A) 催化進行腺苷酸化 (adenylation)反應。然而，目前研究仍對於催化芳香類 α-酮酸 (aromatic α-keto acid) adenlyation反應之 A domain 的受質結合資訊仍不完備。
因此本研究中我們想要闡述 ApvA-A 的受質專一性以及受質辨認機制。 ApvA-A 的基因已透過 cloning 的方式自真菌的 genome 獲得，且純化後之重組蛋白亦具有高純度。分析級的分子篩結果表示 ApvA-A 在水溶液下以二聚體 (dimer)的方式存在。此外，為了了解 ApvA-A 受質專一性，故選擇了 3 種 4-HPPA 的類似物來進行活性比較，分別為 L-Tyr、L-Phe 及 L-DOPA。並透過 hydroxylamine trapping continuous MesG-pyrophosphate 方法來測定酵素活性。結果顯示當受質替換為 L-Tyr、L-DOPA 時，酵素活性均有下降；而當替換為 L-Phe 時則無法偵測到酵素活性。此外，亦嘗試透過蛋白質結晶學欲獲得 ApvA-A 的結構資訊。然而目前並未獲得晶體結構，故後續利用同源性建模方式模擬 ApvA-A 結構資訊，而 ApvA-A 與 4-HPPA 的 complex 模擬結構為透過分子對接 (molecular docking)的方式獲得。於受質結合位中，可見 A216、F260、A333 形成了一個疏水性環境 (hydrophobic surrounding)， 且 4-HPPA 與 F260 產生 π-π stacking 的交互作用。另外，E220 可和 4-HPPA 的 para-hydroxyl group 有氫鍵的形成。上述結果指出 para-hydroxyl group 對於受質辨認扮演著重要角色，且 α-keto group 的有無亦會影響酵素受質辨認。綜上所述，我們藉由酵素活性測定，輔以蛋白質模擬結構的資訊，以對 ApvA-A 的受質辨認機制進行探討。

關鍵字

黑色素 ； 非核糖體肽合成酶 ； 受質專一性 ； 受質辨認 ； 同源性建模

並列摘要

Nonribosomal peptide synthetase (NRPS) is a large multimodular protein category that orchestrates the production of the secondary metabolite containing peptide bonds. Interestingly, the quinone structure-containing natural product could be synthesized by one type of NRPS-like protein, a monomodular protein composed of A-T-TE tri-domain, and had a lack of participation of the C domain in the condensation stage. In Aspergillus terreus, an NRPS-like protein named ApvA is confirmed to synthesize melanin, Aspulvinone E. The A domain in ApvA (ApvA-A) is responsible for adenylating 4-hydroxyphenylpyruvic acid (4-HPPA), an aromatic α-keto acid converted from L-tyrosine by aminotransferase. However, the substrate-binding information of the aromatic α-keto acid adenylated A domain is still poorly understood.
Hence, we aimed to elucidate the substrate specificity and identification of ApvA-A. The gene of ApvA-A was successfully cloned from the fungal genome, and recombinant protein could be produced in high purity. The result of the analytical gel filtration experiment revealed that ApvA-A exists as a dimer form in solution. In addition, various 4-HPPA analogs, including L-Tyr, L-Phe, and L-DOPA, were applied to an activity assay of ApvA-A, which is measured by the hydroxylamine trapping continuous MesG-pyrophosphate method. The activities of L-Tyr and L-DOPA were both lower than that of 4-HPPA, while L-Phe as substrate was not detectable. Furthermore, structural information of ApvA-A was also attempted to approach via both protein crystallography. However, the crystal structure of ApvA-A was not obtained until now. Hence, the ApvA-A model structure was constructed by homology modeling. Furthermore, a complex model of ApvA-A and 4-HPPA was further constructed by using molecular docking, demonstrating that A216, F260, and A333 of ApvA-A could establish a hydrophobic surrounding in the substrate-binding site. The aromatic ring of 4-HPPA could interact with F260 via the π-π stacking. Moreover, E220 could form a hydrogen bond with the hydroxyl group in the para position of 4-HPPA. The result indicated that the para-hydroxyl group in the aromatic ring is crucial to substrate recognition, and the α-keto group would have an influence on substrate identification. In summary, we investigated the substrate identification mechanism of ApvA-A via enzyme activity measurement and homology model information.

並列關鍵字

Melanin ； Nonribosomal peptide synthetase ； Substrate specificity ； Substrate identification ； Homology modeling

參考文獻

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