可抗超级细菌的抗生素——单酰胺菌素（Sulfazecin） 生物合成中羟基化修饰酶的催化机理阐释及其定向改造

The downside of extensive use of antibiotics is the frustrating emergence of renewing drug-resistance, which leads to the rise of superbugs. As one of monobactam antiobitics, sulfazecin remains the few of antibiotics still active against those pathogens. Due to its monocyclic β-lactams structure, sulfazecin is not affected by the drug-resistance genes of superbugs. It is important and helpful to constitute the biosynthesis pathway of sulfazecin for the novel antibiotics design. The hydroxylation modification on the monocyclic β-lactams is required to complete the biosynthesis of sulfazecin. Otherwise, the monocyclic β-lactams intermediates would be hydrolyzed, resulting in the interruption of sulfazecin production. It is proposed that SulO, a non-heme mononuclear iron enzyme, catalyzes the hydroxylation of the monocyclic β-lactams intermediate. However, the problems such as auto-oxidation and low turnover numbers normally exist in such non-heme mononuclear iron enzyme family, which may make SulO a rate-limiting step in the biosynthesis production of sulfazecin. In this proposal, we will explore the catalytic mechanism of SulO reaction, characterize the crystal structure of SulO and also visualize the important residues affecting its activity. Based on the above, we could conduct the directed evolution of SulO to improve its activity and stability, aiming to resolving the rate limiting step issues. This research will pave the way for the biosynthesis production of sulfezecin and its analogues in future.

抗生素的滥用使病菌产生耐药性，导致超级细菌的出现。单酰胺菌素sulfazecin因其特殊的单环结构，不受超级细菌耐药基因的影响，是少数可抵抗超级细菌的抗生素之一。发展单酰胺菌素sulfazecin的生物合成方法将推进可抵抗超级细菌新型抗生素的研发。Sulfazecin生物合成的完成需要在最后两步对单环中间产物进行羟基化修饰。未经修饰的单环中间产物会被合成通路中其他酶元件水解，从而导致sulfazecin生物合成的中断。催化单环中间产物羟基化修饰的酶是所属非血红素单铁酶家族的SulO酶，但这类酶普遍存在自氧化失活、催化循环少的问题，使之成为sulfazecin生物合成途径的限速步骤之一。本研究拟针对sulfazecin生物合成通路中的限速步问题，通过阐明SulO酶催化机理和解析蛋白三维结构，定向改造SulO酶以提升其稳定性和催化活性，解决sulfazencin生物合成中限速步的瓶颈问题。

抗生素的耐药性使现今常用抗生素对超级细菌等耐药菌失效，已经成为迫切需要解决的问题。我们从寻求新型抗生素角度出发，以一种可抗超级细菌抗生素——单酰胺菌素sulfazecin为切入点，通过研究sulfazecin核心四元环结构的生物合成机制，进一步理解新型抗生素的构效关系，发展类似sulfazecin具有较好抗菌抗炎效果的新型抗生素。本项目发展了SulO酶底物及其类似物的有机合成方法，构建了一组SulO酶底物类似物化合物组合。同时针对单酰胺菌素sulfazecin结构中磺酸化修饰，在体外尝试构建sulfazecin的磺酸化引入模块，为发展sulfazecin的类似物结构提供磺酸化生物催化体系。我们进一步针对SulO酶催化机制研究，探究了其可能影响SulO酶催化性质的重要残基，在同源建模的指导下并通过定点突变确认了活性空腔内可参与SulO酶催化的两个络氨酸残基位点，为研究调控SulO酶催化活性并寻求提升SulO酶活性的改造方法提供了新的残基改造靶点和新策略。此外，我们还拓展单酰胺菌素核心结构四元环修饰策略，引入多种不同的修饰基团提升单酰胺菌素四元环结构的抗菌活性，使新型单酰胺菌素四元环结构修饰后具备了广谱的光动力协同抗菌能力，对应用单酰胺菌素核心结构四元环的修饰策略来发展新型和高效可抗超级细菌的单酰胺菌素类抗生素有重要意义。