Stojanoski Vlatko, Chow Dar-Chone, Fryszczyn Bartlomiej, Hu Liya, Nordmann Patrice, Poirel Laurent, Sankaran Banumathi, Prasad B V Venkataram, Palzkill Timothy
§Medical and Molecular Microbiology "Emerging Antibiotic Resistance" Unit, Department of Medicine, Faculty of Science, University of Fribourg, 1700 Fribourg, Switzerland.
∥Berkeley Center for Structural Biology, Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States.
Biochemistry. 2015 Jun 2;54(21):3370-80. doi: 10.1021/acs.biochem.5b00298. Epub 2015 May 14.
OXA-163 and OXA-48 are closely related class D β-lactamases that exhibit different substrate profiles. OXA-163 hydrolyzes oxyimino-cephalosporins, particularly ceftazidime, while OXA-48 prefers carbapenem substrates. OXA-163 differs from OXA-48 by one substitution (S212D) in the active-site β5 strand and a four-amino acid deletion (214-RIEP-217) in the loop connecting the β5 and β6 strands. Although the structure of OXA-48 has been determined, the structure of OXA-163 is unknown. To further understand the basis for their different substrate specificities, we performed enzyme kinetic analysis, inhibition assays, X-ray crystallography, and molecular modeling. The results confirm the carbapenemase nature of OXA-48 and the ability of OXA-163 to hydrolyze the oxyimino-cephalosporin ceftazidime. The crystal structure of OXA-163 determined at 1.72 Å resolution reveals an expanded active site compared to that of OXA-48, which allows the bulky substrate ceftazidime to be accommodated. The structural differences with OXA-48, which cannot hydrolyze ceftazidime, provide a rationale for the change in substrate specificity between the enzymes. OXA-163 also crystallized under another condition that included iodide. The crystal structure determined at 2.87 Å resolution revealed iodide in the active site accompanied by several significant conformational changes, including a distortion of the β5 strand, decarboxylation of Lys73, and distortion of the substrate-binding site. Further studies showed that both OXA-163 and OXA-48 are inhibited in the presence of iodide. In addition, OXA-10, which is not a member of the OXA-48-like family, is also inhibited by iodide. These findings provide a molecular basis for the hydrolysis of ceftazidime by OXA-163 and, more broadly, show how minor sequence changes can profoundly alter the active-site configuration and thereby affect the substrate profile of an enzyme.
OXA-163和OXA-48是密切相关的D类β-内酰胺酶，具有不同的底物谱。OXA-163水解氧亚氨基头孢菌素，特别是头孢他啶，而OXA-48更倾向于碳青霉烯类底物。OXA-163与OXA-48的区别在于活性位点β5链上有一个取代（S212D），以及连接β5和β6链的环中有四个氨基酸缺失（214-RIEP-217）。虽然OXA-48的结构已被确定，但OXA-163的结构尚不清楚。为了进一步了解它们不同底物特异性的基础，我们进行了酶动力学分析、抑制试验、X射线晶体学和分子建模。结果证实了OXA-48的碳青霉烯酶性质以及OXA-163水解氧亚氨基头孢菌素头孢他啶的能力。以1.72 Å分辨率测定的OXA-163晶体结构显示，与OXA-48相比，其活性位点有所扩大，这使得体积较大的底物头孢他啶能够被容纳。与不能水解头孢他啶的OXA-48的结构差异，为这两种酶之间底物特异性的变化提供了一个解释。OXA-163在另一种含有碘化物的条件下也结晶了。以2.87 Å分辨率测定的晶体结构显示，活性位点中有碘化物，同时伴随着几个显著的构象变化，包括β5链的扭曲、Lys73的脱羧以及底物结合位点的扭曲。进一步的研究表明，在碘化物存在的情况下，OXA-163和OXA-48都受到抑制。此外，不属于OXA-48样家族成员的OXA-10也受到碘化物的抑制。这些发现为OXA-163水解头孢他啶提供了分子基础，更广泛地说，展示了微小的序列变化如何深刻地改变活性位点的构型，从而影响酶的底物谱。
