Sapula Sylvia A, Wang Yu, Hart Bradley J, Whittall Jonathan J, Venter Henrietta
UniSA Clinical and Health Sciences, Health and Biomedical Innovation, University of South Australia, Adelaide, SA 5000, Australia.
Biochem J. 2025 Jan 30;482(3):BCJ20240478. doi: 10.1042/BCJ20240478.
The Stenotrophomonas maltophilia L2 cephalosporinase is one of two beta-lactamases that afford S. maltophilia beta-lactam resistance. With the overuse of beta-lactams, selective pressures have contributed to the evolution of these proteins, generating proteins with an extended spectrum of activity. Variant L2 cephalosporinases have been detected, as has their distribution into two main clades (clades 1 and 2). Comprehensive analysis of six L2 variants, cloned into pET41a(+) and expressed in Escherichia coli BL21(DE3) cells, revealed that clade 1 variants exhibited higher ceftazidime resistance compared to clade 2. Notably, the Sm5341 L2 variant, carrying a Phe72Ile variation, displayed a significantly reduced resistance profile across all substrates tested, suggesting a key role of Phe72 in enzymatic activity. An Ile72Phe substitution in the pET41a(+) based Sm5341_L2 variant resulted in a gain-of-function for this protein, confirming the role of Phe72 in the activity of L2. Furthermore, residue interaction network analysis elucidated a pi-cation interaction between Tyr272 and Arg244, which may potentially be stabilizing the enzyme and its binding site. The presence of Tyr272 in clade 1 variants correlates with higher ceftazidime affinity, contrasting Asp272 in clade 2 variants. Displaying lower Km values and higher kcat/Km ratios, clade 1 L2 enzymes demonstrated a higher binding efficiency and greater catalytic efficiency for most of the substrates assessed. These results indicate that L2 enzymes are continuing to evolve and adapt to a selective environment fuelled by the overuse of beta-lactams. This adaptation may signal the beginning of an evolutionary process yielding variant L2 cephalosporinases with extended substrate profiles.
嗜麦芽窄食单胞菌L2头孢菌素酶是赋予嗜麦芽窄食单胞菌β-内酰胺抗性的两种β-内酰胺酶之一。随着β-内酰胺类药物的过度使用，选择性压力促使这些蛋白质发生进化，产生了具有更广活性谱的蛋白质。已检测到L2头孢菌素酶变体，且它们可分为两个主要分支（分支1和分支2）。对克隆到pET41a(+)中并在大肠杆菌BL21(DE3)细胞中表达的6种L2变体进行综合分析发现，与分支2相比，分支1变体对头孢他啶的抗性更高。值得注意的是，携带Phe72Ile变异的Sm5341 L2变体在所有测试底物上的抗性谱均显著降低，这表明Phe72在酶活性中起关键作用。基于pET41a(+)的Sm5341_L2变体中Ile72Phe替换导致该蛋白功能增强，证实了Phe72在L2活性中的作用。此外，残基相互作用网络分析阐明了Tyr272和Arg244之间的π-阳离子相互作用，这可能有助于稳定酶及其结合位点。分支1变体中Tyr272的存在与更高的头孢他啶亲和力相关，而分支2变体中为Asp272。分支1的L2酶显示出更低的Km值和更高的kcat/Km比值，表明在大多数评估底物上具有更高的结合效率和更大的催化效率。这些结果表明，L2酶在持续进化并适应由β-内酰胺类药物过度使用所导致的选择性环境。这种适应性可能标志着一个进化过程的开始，该过程将产生具有更广底物谱的L2头孢菌素酶变体。
