非核醣體胜肽合成酶功能與結構之探討

Abstract

由於抗生素濫用，如今耐藥性細菌不斷地增加使得人們陷入抗生素短缺的問 題。為了尋找更多具有生物活性的分子，科學家們開始對天然物進行更深入的研 究。非核醣體胜肽合成酶(nonribosomal peptide synthetase, NRPS)是一種能夠合 成許多次級代謝物的大型蛋白模組，其所能合成的天然物中多數具有生物活性， 是科學家們目前熱門的研究對象。在這次的研究當中，我們主要著重在非核醣體 胜肽合成酶模組中的 A domain 和 TE domain 上做更進一步的研究。 在非核醣體胜肽合成過程中，A domain 主要的功能是選擇受質和對受質進行 腺苷酸化(adenylation)。為了更深入研究 A domain 對受質的專一性，我們使用演 算法對在 Cy domain 後的 A domain 進行受質預測。由於 Cy domain 會催化雜環化 反應，所以其受質理應要有具有親核力的取代基，如絲胺酸、半胱胺酸和蘇胺酸。 根據這個推論，我們從預測結果中挑選出非上述三者的 A domain 並對其進行研究， 希望能藉此提高預測的準確性。 首先，我們以 EnzChek assay 測試各個 A domain 的活性。有趣的是，在 Me1803 這個模組中，我們觀察到 A domain 對三種胺基酸顯示活性，分別是絲胺 酸、天門冬醯胺和 diaminobutyric acid (Dab)。為了進一步釐清這個發現，我們希 望透過結晶得知其結構。然而，目前晶體的解析度還需要再繼續推進，並且在整 個 Me1803 模組中，有些區域的結構尚未在 X-ray 繞射圖譜上被觀測到。因此，我 們對 Me1803 模組的內容進行調整，希望能拿到更高解析度的晶體。此外，我們 也嘗試使用 LC-MS/MS 作為再進一步確認受質的方法，但並沒有得到確定的結果， 顯示 LC-MS/MS 的監測條件還需要被修正。 在非核醣體胜肽合成中，TE domain 作為其合成路徑中的最後一步，釋放合 成的次級代謝物。多數的 TE domain 是以絲胺酸、天門冬胺酸和組胺酸作為催化 三聯體(catalytic triad)，且對受質的選擇性並不高。然而，在合成多黏菌素 (polymyxin)的 TE domain 中，半胱胺酸取代了絲胺酸作為催化三聯體，並且在 對多黏菌素的環化上具有極高的選擇性。此外，在先前的研究中已被證實，將多黏菌素 B 的 TE domain 其催化三聯體中的半胱胺酸突變為絲胺酸，會導致酵素活 性下降。因此，藉由晶體結構，我們希望能更深入的探討這個特殊的 TE domain。 我們首先測試了以多黏菌素 B 的 TE domain 對以化學方法合成且突變的多黏 菌素 B 受質的活性。結果顯示，在第四個胺基酸突變的多黏菌素 B 受質可以和多 黏菌素 B 的 TE domain 並結合且不被環化釋放。因此，多黏菌素 B 的 TE domain 和於第四個胺基酸突變的受質會一起被送去做共結晶。當然，該 TE domain 本身 也會被送去結晶。 綜上所述，我們呈現了 A domain 和 TE domain 的活性，並嘗試從結構方面去 深入研究這些特殊的非核醣體胜肽合成酶。

The growing crisis of an increasing number of the drug-resistant bacteria, due to the overuse of antibiotics, has become a critical issue nowadays. Overcoming this problem has led to a focus on finding new bioactive molecules. Nonribosomal peptide synthetases (NRPSs) are enzyme assembly lines that produce a diverse range of nature products, including antibiotics and immunosuppressants, showcasing their development potential in the pharmaceutical industry. In this study, we concentrate on two domains: the adenylation (A) domain and the thioesterase (TE) domain in the NRPS module. In the NRPS biosynthetic pathway, the A domain plays a crucial role in selecting and activating the substrate. Algorithmic prediction of NRPS module substrates has been employed for years. Here, we specifically focused on the A domain next to the heterocyclization (Cy) domain. Through in silico predictions, we selected a substrate lacking nucleophilicity for heterocyclic compound formation. To further investigate the substrate of the chosen NRPS modules, BAMF and Me1803, we employed EnzChek assay, crystallization assay and LC-MS/MS detection. Notably, the EnzChek assay revealed that the Me1803 module exhibited activity toward three amino acids: Ser, Asn, and diaminobutyric acid (Dab). To understand this phenomenon, crystallization assay was conducted, though the resolution of the crystal needs to improve, and some structures remain unobservable until now. The Me1803 module contents were modified to enhance crystal quality. In addition, the substrate was identified by the LC/MS-MS, but the result was inclusive. Therefore, optimization of LC/MS-MS conditions is essential for elucidating the details of this NRPS module. The TE domain catalyzes the release of polypeptides in NPRS biosynthesis. In most TE domains, the catalytic triad consists of Ser, Asp and His, and exhibit low substrate selectivity. Different from other TE domains, the polymyxin family deviates from this norm by utilizing Cys instead of Ser in the catalytic triad and demonstrates exceptionally high selectivity in the cyclization of polymyxin. Previous research has proved that mutating Cys to Ser results in reduced enzyme activity. Given the uniqueness of this TE domain, we aim to explore more detailed information through crystal structure analysis. In this study, we tested the activity of the wild-type PmxB TE using mutated synthetic substrates PmxB-N-acetylcysteamine (SNAc). Our observation revealed that the mutation at the fourth Dab could bind onto the TE domain without undergoing cyclization. Consequently, further experiments will involve crystallization of both the PmxB TE itself and the PmxB TE with the PmxB (4-ala)-SNAc substrate to gain additional insights. In summary, we presented the enzyme activity of both the A domain and the TE domain. Furthermore, we endeavored to obtain detailed information about the enzyme’s specificity to the substrate through crystallization.