醣胜肽抗生素生合成酵素蛋白晶體結構及反應機制為基礎的新化學結構生物活性分子設計

Structure- and mechanism-based enzyme design for biologically active new chemical entities on glycopeptide antibiotics

劉祐禎(Yu-Chen Liu)

指導教授 ： 李宗璘

共同指導教授 ： 蔡明道(Ming-Daw Tsai)

國立臺灣大學/生命科學院/生化科學研究所/博士(2013年)

https://doi.org/10.6342/NTU.2013.11171

摘要

細菌抗藥性問題日益嚴重，使得發展新型抗生素更加困難，藉由改變抗生素生合成路徑而改變抗生素結構，提供我們一個可能克服細菌抗藥性問題的機會。Dbv29為參與抗生素A40926生合成之六碳糖氧化酶，利用X光蛋白質結晶學與生物化學的方法，我們解析出Dbv29的蛋白質結構以及催化作用機制，並且發現一對酪胺酸活性基團在輔酶蛋白共價結合、酵素活性及維持蛋白質結構上扮演非常重要的角色。更特別的是，受質在Dbv29與teicoplanin複合體蛋白質結構中以反應中間產物呈現，使我們得以進一步利用Dbv29合成各種不同化學結構的衍生物。在抑菌測試中，部分的衍生物對於具有抗藥性的腸球菌(VRE)比抗生素vancomycin與teicoplanin表現出更好的抑菌效果。因此利用這嶄新的方法我們可以發展出更多不同型態的抗生素衍生物來解決細菌抗藥性問題。S-腺苷甲硫氨酸(SAM)必需之甲基轉移酶(methyltransferases)為最具多樣性及重要生物活性功能的酵素之一。(2S,3S)-β-甲基苯丙氨酸(βMePhe)為非蛋白氨基酸，是組成醣胜肽抗生素mannopeptimycin六環胜肽中的部分化學分子。我們先前的研究發現，mannopeptimycin生合成mppJ基因利用S-腺苷甲硫氨酸(SAM)將苯丙酮酸(Ppy)上苯甲基的碳甲基化而產生β-甲基苯丙酮酸(β-MePpy)，雖然其苯甲基的碳具有部分酸性，但是並不表示這碳原子一定會進行親和性取代反應。特別的是純化之MppJ蛋白質呈現藍綠色，意指MppJ有金屬離子參與催化反應，但不同於目前已知鎂(Mg2+)或鈣(Ca2+)離子必需之甲基轉移酶以及自由基反應機制之甲基轉移酶。我們利用X光蛋白質結晶學與生物化學的方法，解析出MppJ的蛋白質結構以及催化作用機制，藉由分析蛋白質與受質(substrates)及產物(products)複合體蛋白結構，我們發現MppJ為目前唯一鐵離子(Fe3+)及S-腺苷甲硫氨酸(SAM)必需之甲基轉移酶，並且知道MppJ如何在碳原子上發生甲基化反應的催化機制。多個複合體蛋白結構使我們了解MppJ利用鐵離子與α酮基酸(α-ketoacid)結合配位與活化，並且發展出兩個水分子裝置來控制酵素專一性進行碳上甲基化反應。如此發現使我們更進一步將甲基轉移酶改變成結構與功能不相關的新酵素。我們藉由改變金屬離子的配位化學，使得原本酵素變成具有水合酶及甲基轉移酶的雙重功能，進而產生具有立體特異性的新化學分子。

關鍵字

醣胜肽抗生素 ； teicoplanin衍生物 ； 氧化還原酶還原胺化 ； 抗萬古黴素腸球菌(VRE) ； mannopeptimycin ； 非蛋白氨基酸 ； 甲基轉移酶 ； S-腺苷甲硫氨酸(SAM) ； 苯丙酮酸(Ppy)

並列摘要

In the search for new efficacious antibiotics, biosynthetic engineering offers attractive opportunities to introduce minor alterations to antibiotic structures that may overcome resistance. Dbv29, a flavin-containing oxidase, catalyzes the four-electron oxidation of a vancomycin-like glycopeptide to yield A40926. Structural and biochemical examination of Dbv29 now provides insights into residues that govern flavinylation and activity, protein conformation and reaction mechanism. In particular, the serendipitous discovery of a reaction intermediate in the crystal structure led us to identify an unexpected opportunity to intercept the normal enzyme mechanism at two different points to create new teicoplanin analogs. Using this method, we synthesized families of antibiotic analogs with amidated and aminated lipid chains, some of which showed marked potency and efficacy against multidrug resistant pathogens. This method offers a new strategy for the development of chemical diversity to combat antibacterial resistance.
The S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTs) represent one of the most diverse and biologically important classes of enzymes. (2S,3S)-β-methylphenylalanine, a nonproteinogenic amino acid, is a building unit in glycopeptide antibiotic mannopeptimycin. The mppJ gene product in the mannopeptimycin biosynthetic gene cluster was determined to be the committed MT to methylate the benzylic carbon of phenylpyruvate (Ppy) into β-MePpy. The benzylic carbon of Ppy has some extent of acidity, but its acting as an operational nucleophile was not concluded. The purified MppJ displays a turquoise color implicating involvement of a metal ion. The solved crystal structures revealed MppJ the first ferric ion/SAM-dependent MT. Additional four structures in binary and ternary complexes illustrated the molecular mechanism for the metal ion dependent MT reaction. MppJ has evolved a non-heme iron center to bind, orientate and activate the α-ketoacid substrate and meanwhile developed a sandwiched bi-water device to avoid formation of the unwanted reactive oxo-Fe(IV) species during the C-MT reaction. This unprecedented discovery further prompted us to convert the MT into a structurally/functionally unrelated new enzyme. Through stepwise-protein engineering and manipulation of coordination chemistry MTs were engineered to perform both non-heme iron dependent hydration and methyltransferation reactions for stereo-specific new compounds. The process was validated by five crystal structures.

並列關鍵字

glycopeptide antibiotics ； teicoplanin analogs ； oxidoreductase ； reductive amination ； vancomycin-resistant Enterococcus (VRE) ； mannopeptimycin ； nonproteinogenic amino acid ； methyltransferase ； S-adenosyl-L-methionine (SAM) ； phenylpyruvic acid (Ppy)

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