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首页> 外文期刊>Biochemistry >Site-Saturation Mutagenesis of Position V117 in OXA-1 β-Lactamase: Effect of Side Chain Polarity on Enzyme Carboxylation and Substrate Turnover
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Site-Saturation Mutagenesis of Position V117 in OXA-1 β-Lactamase: Effect of Side Chain Polarity on Enzyme Carboxylation and Substrate Turnover

机译：OXA-1β-内酰胺酶中位置V117的位点饱和诱变：侧链极性对酶羧化和底物转换的影响

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Class D β-lactamases pose an emerging threat to the efficacy of β-lactam therapy for bacterial infections. Class D enzymes differ mechanistically from other β-lactamases by the presence of an active-site N-carboxylated lysine that serves as a general base to activate the serine nucleophile for attack. We have used site-saturation muta-genesis at position V117 in the class D β-lactamase OXA-1 to investigate how alterations in the environment around N-carboxylated K70 affect the ability of that modified residue to carry out its normal function. Minimum inhibitory concentration analysis of the 20 position 117 variants demonstrates a clear pattern of charge and polarity effects on the level of ampicillin resistance imparted on Escherichia coli (E. coli). Substitutions that introduce a negative charge (0, E) at position 117 reduce resistance to near background levels, while the positively charged K and R residues maintain the highest resistance levels of all mutants. Treatment of the acidic variants with the fluorescent penicillin BOCILLIN FL followed by SDS-PAGE shows that they are active for acylation by substrate but deacylation-deficient. We used a novel fluorescence anisotropy assay to show that the specific charge and hydrogen-bonding potential of the residue at position 117 affect CO_2 binding to K70, which in turn correlates to deacylation activity. These conclusions are discussed in light of the mechanisms proposed for both class D β-lactamases and BlaR β-lactam sensor proteins and suggest a reason for the preponderance of asparagine at the V117-homologous position in the sensors.
机译：D类β-内酰胺酶对β-内酰胺治疗细菌感染的功效构成了新的威胁。 D类酶在机制上不同于其他β-内酰胺酶，是因为存在活性位点N-羧化赖氨酸，该赖氨酸可作为激活丝氨酸亲核试剂攻击的通用碱基。我们已使用D类内酰胺酶OXA-1中V117处的位点饱和诱变来研究N-羧化K70周围环境的变化如何影响该修饰残基执行其正常功能的能力。对20个位置117变体的最小抑菌浓度分析表明，电荷和极性作用对大肠杆菌（E. coli）的氨苄青霉素抗性水平具有明显的影响。在位置117处引入负电荷（0，E）的取代基将抗性降低至接近本底水平，而带正电的K和R残基则保持所有突变体的最高抗性水平。用荧光青霉素BOCILLIN FL处理酸性变体，然后进行SDS-PAGE显示，它们对被底物酰化具有活性，但缺乏去酰化作用。我们使用一种新型的荧光各向异性测定法来显示，在117位残基的比电荷和氢键势会影响CO_2与K70的结合，进而与脱酰活性相关。根据针对D类β-内酰胺酶和BlaRβ-内酰胺传感器蛋白提出的机制对这些结论进行了讨论，并提出了传感器中V117同源位置上天冬酰胺占优势的原因。

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