具有多重抗生素抗藥性的葛蘭氏陽性菌感染已是眾所關注的公共衛生問題。因此，開發新一代有效對抗抗生素抗藥性病原菌醣胜肽類抗生素有其急迫性。目前為解決抗生素抗藥性問題主要有兩種途徑，一為尋找或創造出新穎性抗生素，二為將現有抗生素修改成更具療效的抗生素衍生物。Teicoplanin (Tei)和A40926這兩種酯醣胜肽類抗生素對於抗甲氧苯青黴素金黃色葡萄球菌的殺菌性優於萬古黴素，而此兩類抗生素不同於萬古黴素主要在於骨架上第四號胺基酸的葡萄糖胺上多了一條十個碳的支鏈。該支鏈能增加藥物的疏水性，延長其藥物的半衰期，及增加殺菌活性。所以在這個研究中，我們試圖以結構和功能為基礎針對參與Tei和A40926生合成的酵素進行蛋白質功能創新及改良。參與Tei和A40926結構中第四號位置長鏈醯基之生合成途徑相關酵素已被確認。其中Orf2*為乙醯水解酶催化乙醯葡萄糖胺上的去乙醯基作用，進而產生親核基讓後續的醯化反應可以進行。本論文嚐試將Tei上第四號位置的乙醯葡萄糖胺長鏈醯基轉換到第六號胺基酸乙醯葡萄糖胺上以創造出更多新穎的酯醣胜肽類抗生素類似物。我們的方式是透過解析出Orf2*-β-d-octyl glucoside和Orf2*-Tei兩個蛋白複合體晶體結構，藉以窺知酵素與受質間的分子辨識及交互作用。
我們首先揭露了先前未知的長鏈醯基結合位置；再經由分子動態模擬分析，我們了解到當第四號和第六號胺基酸之乙醯葡萄糖胺同時存在時，Orf2*主要的反應位置為第四號胺基酸之乙醯葡萄糖胺，因此，為了要讓第六號胺基酸乙醯葡萄糖胺進行脫醯化反應，第四號上的胺基酸乙醯葡萄糖胺必須先被移除。根據此複合體結構解析，我們改良了Orf2*成ㄧS98A/V121A/F193Y三重突變株，其能有效率地脫除第六號胺基酸乙醯葡萄糖胺上的乙醯基。此外, 我們發現來自於A40926生合成途徑中的甲基轉移酶Dbv27可以進行雙重甲基化作用，於是我們利用此一特性來進行保護機化學保護Tei上第一號位置的胺基親核基，最後透過化學合成的方法得以選擇性的在第六號位置的葡萄糖胺上進行長鏈醯基醯化作用。
另一方面，本論文也探討不尋常胺基酸的生合成，因為不尋常胺基酸的生合成對開發新型抗生素有相當的助益。我們對mannopeptimycin骨架中的刍-hydroxyl-enduracididine之生合成感到興趣。參與此生合成途徑的相關酵素已被推論出來為MppO，MppP，MppQ以及MppR。MppO已被證實為一雙氧合酶，其可將enduracididine轉換成刍beta-hydroxyl-enduracididine；MppP和MppQ則被推測為胺基轉基酶，而MppR則為功能未知的蛋白。為了探討enduracididine之生合成途徑，我們首先異源表現出MppP，MppQ以及MppR，結果顯示在大腸桿菌表現宿主中MppP和MppQ為不可溶。因此，我們便先進行MppR蛋白晶體結構的解析，希望藉由結構能推論出它的功能並完整闡釋enduracididine之生合成途徑。在水溶液中MppR結合成為一四聚體蛋白； 比較MppR和K156A突變株活性區電子雲密度圖及質譜蛋白片段分子量的結果，我們發現胺基酸K156以共價鍵的方式與一個結構未知的化合物產生鍵結。從電子雲密度圖的輪廓推論，此未知物可能是enduracididine的前驅物， 所以Lys156應該扮演著一關鍵性的角色，周邊的胺基酸Trp98，Phe116，Glu118，Gln152，His211，Leu215, 及Glu283可能形成分子辨識區及參與催化作用。目前MppR在enduracididine生合成途徑中所扮演的確切角色仍未詳盡，然而對於作進一步的探討本研究已建立良好的基礎。

The emergence of multidrug-resistant Gram-positive pathogens is a serious public health issue. Thus, the development of novel glycopeptide antibiotics with higher efficacy against resistant strains is urgently demanded. Lipoglycopeptide antibiotics, such as teicoplanin (Tei) and A40926, are more effective than vancomycin against methicillin-resistant Staphylococcus aureus (MRSA) in as much as they carry an extra aliphatic acyl side chain on glucosamine (Glm) at residue 4 (r4). In this pursuit, we attempted to reposition N-acyl Glc from r4 to r6 at Tei by structure- and function-based protein engineering. The biosynthesis of the r4 N-acyl Glc moiety at Tei or A40926 has been elucidated, in which the primary amine nucleophile of Glm is freed from the r4 GlcNac pseudo-Tei precursor by Orf2* for the subsequent acylation reaction to occur.
In this dissertation, two Orf2* structures in complex with β-d-octyl glucoside or Tei were solved. Of the complexed structures, the substrate binding site and a previously unknown hydrophobic cavity were revealed, wherein r4 GlcNac acts as the key signature for molecular recognition and the cavity allows substrates carrying longer acyl side chains in addition to the acetyl group. On the basis of the complexed structures, a triple-mutation mutant S98A/V121A/F193Y is able to regioselectively deacetylate r6 GlcNac pseudo-Tei instead of that at r4. On the other hand, a methyltransferase Dbv27 from A40926 biosynthesis pathway was found to be able to double methylate r1 free amine group so that Dbv27 can be used in protection chemistry. Thereby, novel analogs can be selectively made at the r6 sugar moiety through simple organic synthesis.
On the other hand, we are interested in the biosynthesis of nonproteinogenic amino acids because such study is informative in developing new antibiotics. In this study, we explored the biosynthesis of 刍-hydroxy-enduracididine in mannopeptimycin. Four gene products (MppOPQR) were predicted to be involved in the synthesis. The gene products of mppPQ were predicted to be PLP-dependent aminotransferases, while mppR has no functionally known homolog. MppO recently was characterized to be able to hydroxylate enduracididine at the 刍-carbon position. To understand how enduracididine is synthesized, we focus on MppP, MppQ and MppR. MppP and MppQ proteins unfortunately were found to be insoluble in E. coli. Nevertheless, the crystal structures of MppR and the mutant K156A have now been solved. MppR forms as tetramers in solution. Comparing K156A with native MppR, there is no obvious deviation from corresponding residues within the putative active site. In MALDI-MS analysis, the protein fragmentations between the native and K156A MppR suggested that there is an extra moiety covalently bound to Lys156. On the basis of the contour in the density map, this moiety seems to be the precursor of enduracididine. Although the real function of MppR in the biosynthesis of enduracididine remains unknown, this study however has paved a solid foundation for further study in this regard.

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