Tsukamoto K, Ohno R, Sawai T
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
J Bacteriol. 1990 Aug;172(8):4348-51. doi: 10.1128/jb.172.8.4348-4351.1990.
The cephalosporinase of Citrobacter freundii GN346 is a class C beta-lactamase, consisting of 361 amino acids and exhibiting the substrate profile of a typical cephalosporinase. On the conversion of a conserved glutamic acid at residue 219 to lysine, the substrate spectrum of the cephalosporinase was extended to oxyimino cephalosporins, aztreonam and carbenicillin, which are essentially undesirable substrates for the enzyme. Escherichia coli cells carrying the mutant gene showed higher resistance levels to cefuroxime, aztreonam, and carbenicillin, but a lower resistance level to cefoxitin, than cells carrying the wild gene. The kcat values of the purified mutant enzyme for ceftazidime, cefuroxime, and cefmenoxime were 77,100, and 300 times those of the wild enzyme, respectively. The relative Vmax values of the mutant enzyme for aztreonam and carbenicillin were determined to be 11 and 23 times those of the wild enzyme, respectively, but the value of the mutant enzyme for cefoxitin was only one-third that of the wild enzyme.
弗氏柠檬酸杆菌GN346的头孢菌素酶是一种C类β-内酰胺酶，由361个氨基酸组成，具有典型头孢菌素酶的底物谱。当219位保守的谷氨酸残基转变为赖氨酸时，头孢菌素酶的底物谱扩展到氧亚氨基头孢菌素、氨曲南和羧苄青霉素，而这些基本上是该酶不希望有的底物。携带突变基因的大肠杆菌细胞对头孢呋辛、氨曲南和羧苄青霉素表现出更高的耐药水平，但对头孢西丁的耐药水平低于携带野生型基因的细胞。纯化的突变酶对头孢他啶、头孢呋辛和头孢甲肟的kcat值分别是野生型酶的77倍、100倍和300倍。突变酶对氨曲南和羧苄青霉素的相对Vmax值分别确定为野生型酶的11倍和23倍，但突变酶对头孢西丁的值仅为野生型酶的三分之一。
