Structure and dynamics of CTX-M enzymes reveal insights into substrate accommodation by extended-spectrum beta-lactamases.
Oxyimino-cephalosporin antibiotics, such as ceftazidime, escape the hydrolytic activity of most bacterial beta-lactamases. Their widespread use prompted the emergence of the extended-spectrum beta-lactamases CTX-Ms, which have become highly prevalent. The C7 beta-amino thiazol-oxyimino-amide side chain of ceftazidime has a protective effect against most CTX-M beta-lactamases. However, Asp240Gly CTX-M derivatives demonstrate enhanced hydrolytic activity against this compound. In this work, we present the crystallographic structures of Asp240Gly-harboring enzyme CTX-M-16 in complex with ceftazidime-like glycylboronic acid (resolution 1.80 A) and molecular dynamics simulations of the corresponding acyl-enzyme complex. These experiments revealed breathing motions of CTX-M enzymes and the role of the substitution Asp240Gly in the accommodation of ceftazidime. The substitution Asp240Gly resulted in insertion of the C7 beta side chain of ceftazidime deep in the catalytic pocket and orchestrated motions of the active serine Ser70, the beta 3 strand and the omega loop, which favored the key interactions of the residues 237 and 235 with ceftazidime.
Delmas
Julien
J
Laboratoire de Bactériologie, CHU Clermont-Ferrand, Clermont-Ferrand F-63003, France.
Chen
Yu
Y
Prati
Fabio
F
Robin
Frédéric
F
Shoichet
Brian K
BK
Bonnet
Richard
R
D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D001897:Boronic Acids / Q000378:metabolism; D002384:Catalysis; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D005998:Glycine / Q000378:metabolism; D006860:Hydrogen Bonding; D006868:Hydrolysis; D008956:Models, Chemical; D008958:Models, Molecular; D015394:Molecular Structure; D010957:Plasmids; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012399:Rotation; D013379:Substrate Specificity; D014867:Water / Q000737:chemistry; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D001897:Boronic Acids; D014867:Water; C071167:beta-lactamase CTX-2; D001618:beta-Lactamases; D005998:Glycine