结构和功能ArnD. 的结构和功能. 一个变形酶对于脂质A的修改至关重要,具有4-Amino-4-deoxy-l-arabinose和Polymyxin耐药性的4-Amino-4-deoxy-l-arabinose
Daniel Muñoz-Escudero1, Steven D Breazeale2, Myeongseon Lee3
1Department of Molecular Cellular and Developmental Biology, University of Colorado Boulder, Boulder, Colorado 80309, United States.
Biochemistry
|October 2, 2023
概括
确定ArnD是负责去除格兰阴性细菌中的脂质A前体中的甲基基酶的酶. 这一发现阐明了抗生素耐药性机制的一个关键步骤,并揭示了一种新的碳水化合物雌激酶家族.
科学领域:
- 微生物学和分子生物学
- 生物化学和酶学 生物化学和酶学
- 结构生物学 结构生物学
背景情况:
- 格拉姆阴性细菌通过对脂质A与4-脱氧-4-氨基-1-氨酸 (Ara4N) 进行共价修饰,对抗微生物和多素抗生素产生耐药性.
- 生物合成途径涉及ArnC对Ara4N的N-形成,需要下游变形酶来产生活性脂质A前体 (C55P-Ara4N).
研究的目的:
- 为了确定负责Ara4N生物合成途径中变形阶段的特定酶.
- 为了描述已识别的变形酶的结构和功能,ArnD.
主要方法:
- 使用缺乏arnd基因的Escherichia coli突变体进行遗传分析,以观察形式化前体的积累.
- 清洗和生化表征的沙门氏菌 typhimurium ArnD (stArnD). 清洗和生化表征的沙门氏菌 typhimurium ArnD (stArnD). 清洗和生化表征的沙门氏菌 typhimurium ArnD (stArnD).
- 确定stArnD的晶体结构和位点定向突变发生,以探测活性位点残留物.
主要成果:
- 删除arnd导致无二烯--4-脱氧-4-formamido-l-arabinose (C55P-Ara4FN) 的积累,证实了ArnD作为变形酶的作用.
- 星ArnD的晶体结构揭示了一个新的碳水化合物酶家族 (CE4),具有独特的特征,包括膜关联插入.
- stArnD需要双价金属离子 (Co2+或Mn2+) 进行活性,并有效地变形C55P-Ara4FN,突变D9N和H233Y使酶失活.
结论:
- ArnD是Ara4N生物合成途径中必不可少的下游变形酶,对格拉姆阴性细菌的抗生素耐药性至关重要.
- ArnD代表了一种具有独特结构结构的膜相关,金属依赖碳水化合物酶的新型家族.
- 主要活性部位残留物D9和H233对ArnD的催化机制至关重要,可能涉及金属辅助酸催化.
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