位于VIM-2活性部位附近的保存辅助残留物会影响其金属β-乳酸酶活性
Diamond Jain1, Tejavath Ajith1, Jyoti Verma2
1Department of Bioscience and Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur 721302, India.
FEMS microbiology letters
|January 11, 2025
概括
在活性部位附近的维罗纳-整合子-金属-β-乳糖酶 (VIM-2) 中的突变会影响其功能. 替代品N210A和D182A降低了VIM-2的稳定性和催化效率,影响了β-乳酸盐耐药性.
科学领域:
- 微生物学 微生物学
- 生物化学 生物化学
- 分子生物学分子生物学
背景情况:
- 维罗纳整合子金属β-乳糖酶 (VIM-2) 是一种流行的B类β-乳糖酶,对β-乳糖抗生素产生了耐药性.
- 虽然活性部位的残留物对金属结合至关重要,但活性部位附近的残留物也起着重要的功能作用.
研究的目的:
- 调查VIM-2活性部位附近的特定残留物的功能重要性.
- 确定位于E146,D182,N210,S207和D213位置的氨酸替代物对VIM-2活性和稳定性的影响.
主要方法:
- 在体分析中选择关键的遗留物用于突变.
- 局部导向的突变生成,以产生VIM-2的阿兰因替代变体 (VIM-2_E146A,VIM-2_D182A,VIM-2_N210A,VIM-2_S207A,VIM-2_D213A).
- 评估表达野生型和突变VIM-2蛋白质的大肠杆菌中的β-乳酸胺敏感性,然后进行蛋白质净化和催化效率和热稳定性的体外表征.
主要成果:
- VIM-2_N210A增加了宿主对青素和化素的敏感性 (4-8倍).
- VIM-2_D182A显著增加宿主敏感性,在体外催化效率和热稳定性大幅降低.
- VIM-2_E146A的敏感性降低了两倍,触媒效率略有改变,而VIM-2_S207A和VIM-2_D213A的影响在摘要中没有详细说明.
- 发现N210A和D182A突变会对VIM-2的性能产生负面影响,影响基质的特异性和稳定性.
结论:
- 废物N210和D182对VIM-2的功能至关重要,影响其催化效率和稳定性.
- 针对这些近活性场所残留物提供了潜在的策略来对抗VIM-2介导的β-乳酸盐耐药性.
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