EVOLUTION OF AN ENZYME-ACTIVITY - CRYSTALLOGRAPHIC STRUCTURE AT 2-ANGSTROM RESOLUTION OF CEPHALOSPORINASE FROM THE AMPC GENE OF ENTEROBACTER-CLOACAE-P99 AND COMPARISON WITH A CLASS-A PENICILLINASE

被引:206
作者
LOBKOVSKY, E
MOEWS, PC
LIU, HS
ZHAO, HC
FRERE, JM
KNOX, JR
机构
[1] UNIV CONNECTICUT,DEPT MOLEC & CELL BIOL,STORRS,CT 06269
[2] UNIV LIEGE,ENZYMOL LAB,B-4000 SART,BELGIUM
关键词
BETA-LACTAMASE; PROTEIN FOLDING; BACTERIAL BETA-LACTAM RESISTANCE;
D O I
10.1073/pnas.90.23.11257
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
The structure of the class C ampC beta-lactamase (cephalosporinase) from Enterobacter cloacae strain P99 has been established by x-ray crystallography to 2-angstrom resolution and compared to a class A beta-lactamase (penicillinase) structure. The binding site for beta-lactam antibiotics is generally more open than that in penicillinases, in agreement with the ability of the class C beta-lactamases to better bind third-generation cephalosporins. Four corresponding catalytic residues (Ser-64/70, Lys-67/73, Lys-315/234, and Tyr-150/Ser-130 in class C/A) lie in equivalent positions within 0.4 angstrom. Significant differences in positions and accessibilities of Arg-349/244 may explain the inability of clavulanate-type inhibitors to effectively inactivate the class C beta-lactamases. Glu-166, required for deacylation of the beta-lactamoyl intermediate in class A penicillinases, has no counterpart in this cephalosporinase; the nearest candidate, Asp-217, is 10 angstrom from the reactive Ser-64. A comparison of overall tertiary folding shows that the cephalosporinase, more than the penicillinase, is broadly similar to the ancestral beta-lactam-inhibited enzymes of bacterial cell wall synthesis. On this basis, it is proposed that the cephalosporinase is the older of the two beta-lactamases, and, therefore, that a local refolding in the active site, rather than a simple point mutation, was required for the primordial class C beta-lactamase to evolve to the class A beta-lactamase having an improved ability to catalyze the deacylation step of beta-lactam hydrolysis.
引用
收藏
页码:11257 / 11261
页数:5
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