Department of Chemistry & Biochemistry, Texas Tech University, Lubbock, Texas, United States of America.
Department of Cell Physiology & Molecular Biophysics, Texas Tech University Health Sciences Center, Lubbock, Texas, United States of America.
PLoS One. 2019 Apr 22;14(4):e0214440. doi: 10.1371/journal.pone.0214440. eCollection 2019.
The hydrolysis of β-lactam antibiotics by β-lactamase enzymes is the most prominent antibiotic resistance mechanism for many pathogenic bacteria. Out of this broad class of enzymes, metallo-β-lactamases are of special clinical interest because of their broad substrate specificities. Several in vitro inhibitors for various metallo-β-lactamases have been reported with no clinical efficacy. Previously, we described a 10-nucleotide single stranded DNA aptamer (10-mer) that inhibits Bacillus cereus 5/B/6 metallo-β-lactamase very effectively. Here, we find that the aptamer shows uncompetitive inhibition of Bacillus cereus 5/B/6 metallo-β-lactamase during cefuroxime hydrolysis. To understand the mechanism of inhibition, we report a 2.5 Å resolution X-ray crystal structure and solution-state NMR analysis of the free enzyme. Chemical shift perturbations were observed in the HSQC spectra for several residues upon titrating with increasing concentrations of the 10-mer. In the X-ray crystal structure, these residues are distal to the active site, suggesting an allosteric mechanism for the aptamer inhibition of the enzyme. HADDOCK molecular docking simulations suggest that the 10-mer docks 26 Å from the active site. We then mutated the three lysine residues in the basic binding patch to glutamine and measured the catalytic activity and inhibition by the 10-mer. No significant inhibition of these mutants was observed by the 10-mer as compared to wild type. Interestingly, mutation of Lys50 (Lys78; according to standard MBL numbering system) resulted in reduced enzymatic activity relative to wild type in the absence of inhibitor, further highlighting an allosteric mechanism for inhibition.
β-内酰胺酶对β-内酰胺类抗生素的水解是许多病原菌对抗生素产生耐药性的最主要机制。在这一大类酶中，金属β-内酰胺酶因其广泛的底物特异性而具有特殊的临床意义。已经报道了几种针对各种金属β-内酰胺酶的体外抑制剂，但没有临床疗效。以前，我们描述了一种 10 个核苷酸的单链 DNA 适体（10mer），它能非常有效地抑制蜡样芽孢杆菌 5/B/6 金属β-内酰胺酶。在这里，我们发现该适体在头孢呋辛水解过程中对蜡样芽孢杆菌 5/B/6 金属β-内酰胺酶表现出非竞争性抑制。为了了解抑制机制，我们报道了该游离酶的 2.5Å分辨率 X 射线晶体结构和溶液态 NMR 分析。随着 10mer 浓度的增加，HSQC 谱中观察到几个残基的化学位移扰动。在 X 射线晶体结构中，这些残基远离活性位点，这表明适体对酶的抑制作用是一种变构机制。HADDOCK 分子对接模拟表明，10mer 从活性位点对接 26Å。然后，我们将碱性结合斑中的三个赖氨酸残基突变为谷氨酰胺，并测量了催化活性和 10mer 的抑制作用。与野生型相比，10mer 对这些突变体没有明显的抑制作用。有趣的是，与野生型相比，在没有抑制剂的情况下，Lys50（根据标准 MBL 编号系统为 Lys78）的突变导致酶活性降低，这进一步突出了抑制的变构机制。
