Tamaki M, Nukaga M, Sawai T
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Biochemistry. 1994 Aug 23;33(33):10200-6. doi: 10.1021/bi00199a049.
The chromosomal beta-lactamase gene of Proteus vulgaris K1 was cloned and sequenced. The gene comprises 813 nucleotides and codes for the mature enzyme of 29,655 Da, comprising 271 amino acids. The K1 beta-lactamase showed 30-70% similarity, in the overall amino acid sequence, to class A beta-lactamases of Gram-negative bacteria. However, the K1 beta-lactamase differs from most class A enzymes in having a unique substrate specificity as a cephalosporinase, its spectrum extending to even oxyiminocephalosporins. To clarify the relationship between its unique substrate specificity and specific amino acid residues, alignment of the amino acid sequence of the K1 beta-lactamase with those of class A beta-lactamases was performed, and Ala104 and Ser237 were found to be candidates. Ala104 and Ser237 were replaced with glutamic acid and alanine, respectively, which are commonly found in other class A beta-lactamases. The substitution at position 104 had no effect on the enzyme activity or the substrate specificity. The amino acid replacement at position 237, however, reduced the kcat/Km value for an oxyiminocephalosporin (cefuroxime) to 17% of that in the case of the wild-type enzyme, whereas the mutant enzyme showed a higher kcat/Km value for benzylpenicillin, 3 times, than that of the wild-type enzyme. These results indicated that Ser237 is one of the residues responsible for the unique substrate specificity of the P. vulgaris beta-lactamase.
普通变形杆菌K1的染色体β-内酰胺酶基因被克隆并测序。该基因由813个核苷酸组成，编码分子量为29,655 Da的成熟酶，包含271个氨基酸。K1β-内酰胺酶在整体氨基酸序列上与革兰氏阴性菌的A类β-内酰胺酶有30%-70%的相似性。然而，K1β-内酰胺酶与大多数A类酶不同，它作为头孢菌素酶具有独特的底物特异性，其作用谱甚至延伸到氧亚氨基头孢菌素。为了阐明其独特底物特异性与特定氨基酸残基之间的关系，将K1β-内酰胺酶的氨基酸序列与A类β-内酰胺酶的序列进行比对，发现Ala104和Ser237是候选位点。分别用其他A类β-内酰胺酶中常见的谷氨酸和丙氨酸替换Ala104和Ser237。104位的替换对酶活性和底物特异性没有影响。然而，237位的氨基酸替换使氧亚氨基头孢菌素（头孢呋辛）的kcat/Km值降至野生型酶的17%，而突变酶对苄青霉素的kcat/Km值比野生型酶高3倍。这些结果表明，Ser237是普通变形杆菌β-内酰胺酶独特底物特异性的关键残基之一。
