Cao Thinh-Phat, Yi Hyojeong, Dhanasingh Immanuel, Ghosh Suparna, Choi Jin Myung, Lee Kun Ho, Ryu Seol, Kim Heenam Stanley, Lee Sung Haeng
Department of Cellular and Molecular Medicine, Chosun University School of Medicine, Gwangju, South Korea.
Department of Biomedical Sciences, Gwangju Alzheimer's Disease and Related Dementia Cohort Research Center, College of Natural Sciences and Public Health and Safety, Chosun University, Gwangju, South Korea.
Front Mol Biosci. 2020 Nov 27;7:598998. doi: 10.3389/fmolb.2020.598998. eCollection 2020.
Despite class A ESBLs carrying substitutions outside catalytic regions, such as Cys69Tyr or Asn136Asp, have emerged as new clinical threats, the molecular mechanisms underlying their acquired antibiotics-hydrolytic activity remains unclear. We discovered that this non-catalytic-region (NCR) mutations induce significant dislocation of β3-β4 strands, conformational changes in critical residues associated with ligand binding to the lid domain, dynamic fluctuation of Ω-loop and β3-β4 elements. Such structural changes increase catalytic regions' flexibility, enlarge active site, and thereby accommodate third-generation cephalosporin antibiotics, ceftazidime (CAZ). Notably, the electrostatic property around the oxyanion hole of Cys69Tyr ESBL is significantly changed, resulting in possible additional stabilization of the acyl-enzyme intermediate. Interestingly, the NCR mutations are as effective for antibiotic resistance by altering the structure and dynamics in regions mediating substrate recognition and binding as single amino-acid substitutions in the catalytic region of the canonical ESBLs. We believe that our findings are crucial in developing successful therapeutic strategies against diverse class A ESBLs, including the new NCR-ESBLs.
尽管携带催化区域外替换（如Cys69Tyr或Asn136Asp）的A类超广谱β-内酰胺酶（ESBLs）已成为新的临床威胁，但其获得性抗生素水解活性的分子机制仍不清楚。我们发现，这种非催化区域（NCR）突变会导致β3-β4链显著错位、与配体结合到盖子结构域相关的关键残基构象变化、Ω环和β3-β4元件的动态波动。这些结构变化增加了催化区域的灵活性，扩大了活性位点，从而容纳第三代头孢菌素抗生素头孢他啶（CAZ）。值得注意的是，Cys69Tyr ESBL的氧负离子孔周围的静电性质发生了显著变化，导致酰基酶中间体可能得到额外的稳定。有趣的是，NCR突变通过改变介导底物识别和结合区域的结构和动力学，与经典ESBLs催化区域中的单氨基酸替换一样，对抗生素耐药性有效。我们相信，我们的发现对于制定针对包括新型NCR-ESBLs在内的多种A类ESBLs的成功治疗策略至关重要。
