| 摘要: | 嘉賜黴素(Kasugamycin, KSM)是一種由 Streptomyces kasugiensis M-338 分離出具生物活性之胺基糖苷類抗生素，於 1965 年被發現。其化學結構由甘氨酸亞胺(glycine imine)、kasugamine 和 D-右旋肌醇(D-chiro-inositol)所組成。KSM 迄今已有多種生物活性被報導。首先，對於缺乏對細菌性疾病的基因抗性之農作物，KSM 主要用於預防蘋果和梨樹的火疫病。此外，有研究表明，包括 KSM 在內的胺基糖苷類藥物在局部給予後，能提升鼻黏膜對抗 HSV-2(herpes simplex virus-2)和流感 A 病毒的效果。近期更發現，類幾丁質酶（chitinase 3-like-1 ,CHI3L1）能增強上皮細胞對表達 α、β、γ、δ 或 omicron S 蛋白的偽病毒的感染，而 CHI3L1 的抑制劑與 KSM 則可抑制這些變異株偽病毒對上皮細胞的感染。學者 Ikeno 等人於 2006 年發表了關於嘉賜黴素(KSM)的生合成基因群研究，其中包含 20 個開放性閱讀框架，然而，各個基因在嘉賜黴素生合成路徑中的確切角色仍然需要實驗來釐清。在研究結果中，我們成功表現與純化 KSM 生合成路徑中的 5 個蛋白 KasQ、KasD、KasP、KasR 和 KasC，並嘗試以 HPLC 搭配質譜儀來分析其酵素活性。我們利用 NahK、BLUSP、及 PpA1 以生物合成和化學合成製備無法經由商購取得之數個推測受質提供 KasD 酵素功能的研究，根據實驗結果推測，KasQ 的產物 UDP-ManNAc 並非 KasD 的受質而是 UDP-mannose 或 UDP-mannosamine。另外我們也成功解析 KasR 與 KasH 之蛋白質結構，透過結構解析與單點突變測試，來進一步探討反應機制與拓展這些
酵素功能上的運用。;Kasugamycin (KSM), a bioactive aminoglycoside antibiotic, was isolated from Streptomyces kasugiensis M-338 in 1965. It is made of three chemical entities glycine imine, kasugamine, and D-chiro-inositol. KSM is used to containing bacterial diseases in crops lacking genetic resistance, for example, blight in apple and pear trees. Topical application of aminoglycoside antibiotics, including KSM, has been shown effectiveness against herpes simplex virus-2 (HSV-2) and influenza A virus infections in nasal mucosa. Recent research further revealed that chitinase 3-like-1 (CHI3L1) enhances epithelial cell infection of pseudoviruses expressing various spike proteins, while this process can be repressed by anti CHI3L1 or KSM. In 2006, Ikeno and colleagues identified the biosynthetic gene cluster (BGC) of KSM, which is comprised of 20 open reading frames (ORFs). However, the biochemical role of each individual gene in the KSM BCG still remains undetermined in vitro and/or in vivo. In the elucidation of KSM biosynthesis, I have examined and purified five ORFs from the KSM BGC: KasQ, KasD, KasP, KasR, and KasC. These purified enzymes were subjected to enzymatic examinations and HPLC-MS analyses in a hope to pinpoint their biological functions. KasD predicted to be 4-keo-6-dehydratase was assayed in the first place as it may be an initial enzyme. Because one can’t make bricks without straw, three putative substrates of KasD UDP ManNAc, UDP-mannose and UDP-mannosamine, which are not commercially available, were prepared by means of organic synthesis in conjunction with biocatalysts from the Leloir biosynthetic pathway. Based on our experimental results, we ruled out UDP-ManNAc as a possible substrate for KasD, thus underscoring UDP-mannose or UDP-mannosamine the likely candidate. In the meanwhile, we set up crystallization screening for these proteins, particularly, KasR and KasH, in a way to understand their reaction mechanisms at molecular level. Up to now, several KasH mutants have been made, which should allow us to explore substrate tolerance as well as establish new functionalities for future studies and utilizations. |
