Structure and Dynamics of CTX-M Enzymes Reveal Insights into Substrate Accommodation by Extended-spectrum β-Lactamases Julien Delmas 1,2 , Yu Chen 3 , Fabio Prati 4 , Frédéric Robin 1,2 , Brian K. Shoichet 3 and Richard Bonnet 1,2 ⁎ 1 Laboratoire de Bactériologie, CHU Clermont-Ferrand, Clermont-Ferrand F-63003, France 2 Laboratoire de Bactériologie, UFR Médecine, Université Clermont1, EA3844, Clermont-Ferrand F-63001, France 3 Department of Pharmaceutical Chemistry, University of California, San Francisco, QB3 Building Room 508D, 1700 4th Street, San Francisco, California 94143-2550, USA 4 Department of Chemistry, Università di Modena e Reggio Emilia, via Campi 183, Modena, Italy Received 8 August 2007; received in revised form 8 October 2007; accepted 10 October 2007 Available online 16 October 2007 Oxyimino-cephalosporin antibiotics, such as ceftazidime, escape the hydrolytic activity of most bacterial β-lactamases. Their widespread use prompted the emergence of the extended-spectrum β-lactamases CTX-Ms, which have become highly prevalent. The C7 β-amino thiazol-oxyimino- amide side chain of ceftazidime has a protective effect against most CTX-M β-lactamases. However, Asp240Gly CTX-M derivatives demonstrate en- hanced 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 Å) and molecular dynamics simulations of the corresponding acyl–enzyme com- plex. 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 C7β side chain of ceftazidime deep in the catalytic pocket and orchestrated motions of the active serine Ser70, the β3 strand and the omega loop, which favored the key interactions of the residues 237 and 235 with ceftazidime. © 2007 Elsevier Ltd. All rights reserved. Edited by M. Guss Keywords: β-lactamases; ceftazidime; CTX-M; spectrum; structure Introduction The production of β-lactamases is the predomi- nant cause of resistance to β-lactam antibiotics in Gram-negative bacteria. Oxyimino-cephalosporins such as ceftazidime and cefotaxime escape the hydrolytic activity of most β-lactamases. These β- lactams harbor bulky C7β aminothiazol-oxyi- mino-amide side chains that make them inher- ently less susceptible to β-lactamases. The methyl function of the cefotaxime oxyimino group is replaced by a carboxypropyl function in ceftazi- dime (Fig. 1a). Unfortunately, their widespread use prompted the emergence of extended-spec- trum β-lactamases (ESBLs). These enzymes confer resistance to oxyimino-cephalosporins and lead to poor clinical outcomes. Before 2000, most ESBLs were derived from penicillinases, such as TEM-1 or SHV-1, by point substitutions. In these ESBLs, the substitutions displace two major walls of catalytic pocket, the β3 strand or the omega loop, and lead to an enlarged active site, which is able to recognize the large aminothiazol-oxyimino- cephalosporins. 1,2 *Corresponding author. E-mail address: rbonnet@chu-clermontferrand.fr. Abbreviations used: ESBL, extended-spectrum β-lactamase; MDS, molecular dynamics simulation. doi:10.1016/j.jmb.2007.10.026 J. Mol. Biol. (2008) 375, 192–201 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.