Kinetic and crystallographic studies of extended-spectrum GES-11, GES-12, and GES-14 β-lactamases.
GES-1 is a class A extended-spectrum β-lactamase conferring resistance to penicillins, narrow- and expanded-spectrum cephalosporins, and ceftazidime. However, GES-1 poorly hydrolyzes aztreonam and cephamycins and exhibits very low k(cat) values for carbapenems. Twenty-two GES variants have been discovered thus far, differing from each other by 1 to 3 amino acid substitutions that affect substrate specificity. GES-11 possesses a Gly243Ala substitution which seems to confer to this variant an increased activity against aztreonam and ceftazidime. GES-12 differs from GES-11 by a single Thr237Ala substitution, while GES-14 differs from GES-11 by the Gly170Ser mutation, which is known to confer increased carbapenemase activity. GES-11 and GES-12 were kinetically characterized and compared to GES-1 and GES-14. Purified GES-11 and GES-12 showed strong activities against most tested β-lactams, with the exception of temocillin, cefoxitin, and carbapenems. Both variants showed a significantly increased rate of hydrolysis of cefotaxime, ceftazidime, and aztreonam. On the other hand, GES-11 and GES-12 (and GES-14) variants all containing Ala243 exhibited increased susceptibility to classical inhibitors. The crystallographic structures of the GES-11 and GES-14 β-lactamases were solved. The overall structures of GES-11 and GES-14 are similar to that of GES-1. The Gly243Ala substitution caused only subtle local rearrangements, notably in the typical carbapenemase disulfide bond. The active sites of GES-14 and GES-11 are very similar, with the Gly170Ser substitution leading only to the formation of additional hydrogen bonds of the Ser residue with hydrolytic water and the Glu166 residue.
Institute of Molecular Biotechnology, RWTH-Aachen University, Aachen, Germany.
Heinrich
H
Delbrück
Pierre
P
Bogaerts
Michaël B
MB
Kupper
Roberta
R
Rezende de Castro
Sandra
S
Bennink
Youri
Y
Glupczynski
Moreno
M
Galleni
Kurt M
KM
Hoffmann
Carine
C
Bebrone
D040981:Acinetobacter baumannii / Q000737:chemistry* / Q000201:enzymology; D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000737:chemistry*; D015780:Carbapenems / Q000737:chemistry*; D002511:Cephalosporins / Q000737:chemistry*; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D013329:Structure-Activity Relationship; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics; D047090:beta-Lactams / Q000737:chemistry*
D000900:Anti-Bacterial Agents; D015780:Carbapenems; D002511:Cephalosporins; D007527:Isoenzymes; D011994:Recombinant Proteins; D065093:beta-Lactamase Inhibitors; D047090:beta-Lactams; C405902:GES-1 beta-lactamase; D001618:beta-Lactamases