Role of Asp104 in the SHV β-lactamase
Role of Asp104 in the SHV β-lactamase
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DOI：
10.1128/aac.00848-06
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发表时间：
2006-12-01
影响因子：
4.9
通讯作者：
Bonomo, Robert A.
中科院分区：
文献类型：
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作者：
Bethel, Christopher R.;Hujer, Andrea M.;Bonomo, Robert A.
在TEM型超广谱β-内酰胺酶（ESBL）中，当发现有其他取代时，Ambler位置104处的氨基酸变化（Glu至Lys）导致对头孢他啶和头孢噻肟的抗性增加（例如，Gly 238 Ser和Arg 164 Ser）。为了检测Asp 104在SHV β-内酰胺酶中的作用，进行了位点饱和诱变。我们的目标是调查在这个位置上的氨基酸残基的性质，影响青霉素和氧亚氨基头孢菌素的耐药性。出乎意料的是，在大肠杆菌DH 10 B中表达的SHV中位置104处的58%的氨基酸变体导致对氨苄青霉素的抗性降低的β-内酰胺酶。相反，仅Asp 104 Arg和Asp 104 Lys β-内酰胺酶对头孢噻肟耐药性增加。当所有19个取代被引入SHV-2（Gly 238 Ser）ESBL时，对于双取代的Asp 104 Lys Gly 238 Ser和双取代的Asp 104 Arg Gly 238 Ser β-内酰胺酶，头孢噻肟和头孢他啶耐药性的增加最显著。相应地，头孢噻肟水解的总催化效率（k（cat）/K-m）从Gly 238 Ser的0.60 +/- 0.07 μ M-1 s（-1）（平均值+/-标准差）增加到Asp 104 Lys和Gly 238 Ser β-内酰胺酶的1.70 +/- 0.01 μ M-1 s（-1）（增加三倍）。我们还发现，（i）k（3）是Asp 104 Lys水解头孢噻肟的限速步骤，（ii）随着pH值的增加，双取代Asp 104 Lys Gly 238 Ser变体的头孢噻肟的Km接近Gly 238 Ser β-内酰胺酶的Km，和（iii）104位的Lys与Gly 238 Ser取代以能量加和的方式起作用，以增强头孢噻吩的催化作用。基于这一分析，我们提出，在SHV-1 β-内酰胺酶的Ambler位置104的氨基酸起着重要的作用，在底物结合和识别的氧亚氨基头孢菌素和影响的相互作用Tyr 105与青霉素。
Among the TEM-type extended-spectrum beta-lactamases (ESBLs), an amino acid change at Ambler position 104 (Glu to Lys) results in increased resistance to ceftazidime and cefotaxime when found with other substitutions (e.g., Gly238Ser and Arg164Ser). To examine the role of Asp104 in SHV beta-lactamases, site saturation mutagenesis was performed. Our goal was to investigate the properties of amino acid residues at this position that affect resistance to penicillins and oxyimino-cephalosporins. Unexpectedly, 58% of amino acid variants at position 104 in SHV expressed in Escherichia coli DH10B resulted in beta-lactamases with lowered resistance to ampicillin. In contrast, increased resistance to cefotaxime was demonstrated only for the Asp104Arg and Asp104Lys beta-lactamases. When all 19 substitutions were introduced into the SHV-2 (Gly238Ser) ESBL, the most significant increases in cefotaxime and ceftazidime resistance were noted for both the doubly substituted Asp104Lys Gly238Ser and the doubly substituted Asp104Arg Gly238Ser beta-lactamases. Correspondingly, the overall catalytic efficiency (k(cat)/K-m) of hydrolysis for cefotaxime was increased from 0.60 +/- 0.07 mu M-1 s(-1) (mean +/- standard deviation) for Gly238Ser to 1.70 +/- 0.01 mu M-1 s(-1) for the Asp104Lys and Gly238Ser beta-lactamase (threefold increase). We also showed that (i) k(3) was the rate-limiting step for the hydrolysis of cefotaxime by Asp104Lys, (ii) the K-m for cefotaxime of the doubly substituted Asp104Lys Gly238Ser variant approached that of the Gly238Ser beta-lactamase as pH increased, and (iii) Lys at position 104 functions in an energetically additive manner with the Gly238Ser substitution to enhance catalysis of cephalothin. Based on this analysis, we propose that the amino acid at Ambler position 104 in SHV-1 beta-lactamase plays a major role in substrate binding and recognition of oxyimino-cephalosporins and influences the interactions of Tyr105 with penicillins.