Nukaga M, Tanimoto K, Tsukamoto K, Imajo S, Ishiguro M, Sawai T
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
FEBS Lett. 1993 Oct 11;332(1-2):93-8. doi: 10.1016/0014-5793(93)80491-c.
The class C beta-lactamase of Citrobacter freundii GN346 is a typical cephalosporinase comprising 361 amino acids. The aspartic acid at position 217 and glutamic acid at position 219 in this beta-lactamase were, respectively, previously shown not to be the counterpart of Glu166 (ABL166) in class A beta-lactamases, even though sequence alignment of class A and C enzymes strongly suggested this possibility [(1990) FEBS Lett. 264, 211-214; (1990) J. Bacteriol. 172, 4348-4351]. We tried again to assign candidates for the counterpart of Glu166 through sequence alignment based on other criteria, the glutamic acids at positions 195 and 205 in the class C beta-lactamase being selected. To investigate this possibility, these two glutamic acids were changed to glutamine, lysine or alanine, respectively. All the mutant enzymes showed more than 50% of the activity of the wild-type enzyme, indicating that the possibility was ruled out. These results strongly suggested the possibility that the class C beta-lactamase lacks a functional acidic residue corresponding to Glu166 in class A enzymes.
弗氏柠檬酸杆菌GN346的C类β-内酰胺酶是一种典型的头孢菌素酶，由361个氨基酸组成。此前研究表明，该β-内酰胺酶第217位的天冬氨酸和第219位的谷氨酸并非A类β-内酰胺酶中Glu166（ABL166）的对应残基，尽管A类和C类酶的序列比对强烈暗示了这种可能性[（1990年）《欧洲生物化学学会联合会快报》264卷，211 - 214页；（1990年）《细菌学杂志》172卷，4348 - 4351页]。我们再次尝试根据其他标准通过序列比对来确定Glu166对应残基的候选者，选择了C类β-内酰胺酶中第195位和第205位的谷氨酸。为研究这种可能性，分别将这两个谷氨酸突变为谷氨酰胺、赖氨酸或丙氨酸。所有突变酶的活性均超过野生型酶的50%，这表明这种可能性被排除。这些结果有力地表明，C类β-内酰胺酶可能缺乏与A类酶中Glu166相对应的功能性酸性残基。
