Amino acid sequence determinants of beta-lactamase structure and activity
Amino acid sequence determinants of beta-lactamase structure and activity
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DOI：
10.1006/jmbi.1996.0279
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发表时间：
1996-05-17
影响因子：
5.6
通讯作者：
Palzkill, T
中科院分区：
文献类型：
--
作者：
Huang, WZ;Petrosino, J;Palzkill, T
TEM-1 β -内酰胺酶催化-内酰胺类抗生素如青霉素类和头孢菌素类的水解，从而使细菌对这些化合物产生耐药性。为了确定对TEM-1 β -内酰胺酶的结构和功能至关重要的氨基酸残基，使用位点定向诱变程序对构成该酶成熟形式的263个氨基酸残基的每个密码子进行随机化。选择功能性随机突变体是基于它们对大肠杆菌产生氨苄西林耐药性的能力。对每组随机突变体测定若干功能突变体的DNA序列。结果发现，263个氨基酸残基中有43个不能耐受取代，因此对酶的结构和活性至关重要。此外，将功能性β -内酰胺酶突变体中的保守残基位置与β -内酰胺酶基因家族中的保守残基位置进行比较，发现许多位置在诱变研究中不能耐受替换，但在基因家族成员中可以自由替换。这一观察结果可能是由于基因家族成员之间补偿突变的积累。最后，利用信息理论熵计算每个位置的有效替换数，量化功能突变体中残基位置上的序列可变性：这些值用于定量估计残基位置上的序列可变性与残基可达表面积分数之间的相关性。这种相关性在统计上是显著的，因为埋藏的残留物往往表现出低变异性，而不变的残留物往往表现出低溶剂暴露。然而，相关性很弱，因为大多数残基既不是完全埋藏的，也不是不变的。(C) 1996学术出版社有限公司
TEM-1 beta-lactamase catalyzes the hydrolysis of beta-lactam antibiotics such as the penicillins and cephalosporins, thus providing for bacterial resistance to these compounds. To determine the amino acid residues critical for the structure and function of TEM-1 beta-lactamase, the codons for each of the 263 amino acid residues that constitute the mature form of the enzyme were randomized using a site-directed mutagenesis procedure. Functional random mutants were selected based on their ability to confer ampicillin resistance to Escherichia coli. The DNA sequence of several functional mutants was determined for each set of random mutants. It was found that 43 out of the 263 amino acid residues do not tolerate substitutions and therefore are critical for the structure and activity of the enzyme. In addition, a comparison of conserved residue positions among functional beta-lactamase mutants with conserved residues in the beta-lactamase gene family identified many positions which did not tolerate substitutions in the mutagenesis studies but are freely substituted among members of the gene family This observation may be due to the accumulation of compensating mutations among members of the gene family. Finally, the sequence variability at residue positions among functional mutants was quantitated by calculating the effective number of substitutions at each position using information-theoretical entropy: These values were used to obtain a quantitative estimate of the correlation between the sequence variability at a position and the fractional accessible surface area of the residue. The correlation is found to be statistically significant in that buried residues tend to exhibit low variability and invariant residues tend to exhibit low solvent exposure. However, the correlation is weak because most residues are neither completely buried nor invariant. (C) 1996 Academic Press Limited