Structure of Apo- and Monometalated Forms of NDM-1—A Highly Potent Carbapenem-Hydrolyzing Metallo-β-Lactamase

et al. (2011) Structure of Apo- and Monometalated Forms of NDM-1-A Highly Potent Carbapenem- Hydrolyzing Metallo-b-Lactamase. PLoS ONE 6(9): e24621. doi:10.1371/journal.pone.0024621 Structure of Apo- and Monometalated Forms of NDM- 1-A Highly Potent Carbapenem-Hydrolyzing Metallo-b- Lactamase Youngchang Kim 0 Christine Tesar 0 Joseph Mire 0 Robert Jedrzejczak 0 Andrew Binkowski 0 Gyorgy 0 Babnigg 0 James Sacchettini 0 Andrzej Joachimiak 0 Adam Driks, Loyola University Medical Center, United States of America 0 1 Midwest Center for Structural Genomics and Structural Biology Center , Biosciences , Argonne National Laboratory, Argonne, Illinois, United States of America, 2 Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, United States of America, 3 The University of Chicago, Department of Molecular Genetics & Cell Biology, Chicago, Illinois, United States of America , 4 Argonne, Illinois , United States of America The New Delhi Metallo-b-lactamase (NDM-1) gene makes multiple pathogenic microorganisms resistant to all known blactam antibiotics. The rapid emergence of NDM-1 has been linked to mobile plasmids that move between different strains resulting in world-wide dissemination. Biochemical studies revealed that NDM-1 is capable of efficiently hydrolyzing a wide range of b-lactams, including many carbapenems considered as ''last resort'' antibiotics. The crystal structures of metal-free apo- and monozinc forms of NDM-1 presented here revealed an enlarged and flexible active site of class B1 metallo-blactamase. This site is capable of accommodating many b-lactam substrates by having many of the catalytic residues on flexible loops, which explains the observed extended spectrum activity of this zinc dependent b-lactamase. Indeed, five loops contribute ''keg'' residues in the active site including side chains involved in metal binding. Loop 1 in particular, shows conformational flexibility, apparently related to the acceptance and positioning of substrates for cleavage by a zincactivated water molecule. - Funding: This work was supported by National Institutes of Health grant GM094585 (AJ), GM094568 (JS) and by the U. S. Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. The imminent threat posed by the recent discovery and dissemination of the plasmid encoded New Delhi Metallo-blactamase (NDM-1) gene (blaNDM-1) harbored by multiple pathogenic microorganisms has prompted the formation of a global scientific corps darmee [1]. Biochemical and structural elucidation of NDM-1 facilitates the thorough mechanistic understanding required for a rational design of small molecule inhibitors specific to NDM-1 for co-administration with b-lactam antibiotics. The crystal structures of NDM-1 presented here reveal an open, enlarged and flexible active site that explains the observed extended spectrum activity of this zinc dependent b-lactamase. One of the last lines of defense against multiple and extensively drug resistant infections is the carbapenem class of b-lactam antibiotics, which was developed to evade b-lactamase mediated resistance posed by aerobic as well as anaerobic pathogens. Unfortunately, the integrity of the big guns (meropenem, imipenem, doripenem, ertapenem) has become compromised by a number of b-lactamases with extended spectrum activity, that is, the ability to inactivate all classes of b-lactam antibiotics, including carbapenems [2]. b-Lactams are the most broadly used antibacterials world-wide due to their effectiveness at irreversibly inhibiting cell wall biosynthetic enzymes required for peptidoglycan recycling, and minimal toxicity in humans [3,4]. The first b-lactam discovered penicillininhibits the function of the D-Ala-D-Ala transpeptidase that links the peptidoglycan molecules in bacteria [4]. Simultaneously, cell wall hydrolases and autolysins continue to break down peptidoglycan crosslinks, resulting in cellular lysis and death. Since the discovery of penicillin, several classes of naturally occurring and semi-synthetic b-lactams have entered the clinic. Concomitantly, broad use of b-lactams as antibacterials applies a selective pressure that increases the reproductive success of pathogenic strains carrying evolved b-lactamase genes capable of combating our arsenal of b-lactam antibiotics. The vast structural diversity designed into the semi-synthetic blactams evades b-lactamase mediated resistance by either preventing initial Michaelis complex formation, or by stabilizing transient intermediates that inhibit further b-lactam turnover. Carbapenems have proven to be the most effective broadspectrum b-lactams, and their utility is generally reserved as a last line of defense against the toughest drug-resistant infections including MRSA [5], XDR-TB [6], and bacterial meningitis [7]. However, it appears that the target met the challenge. In the past several years, new pathogenic strains carrying carbapenemase genes have been documented in patients from India, Pakistan, Bangladesh and other countries [8,9,10,11,12,13,14]. Carbapenemases are members of class A (KPC, IMI/NMC, SME), class B (IMP, VIM, SPM) and D (OXA) b-lactamases, for review see references [15,16,17]. Class B b-lactamases depend on divalent cation metal cofactors for their activity, and are described as metallo-b-lactamases (MBLs) [15,17,18,19]. Unlike serine blactamases, MBLs are not inhibited by the classic irreversible blactamase inhibitors such as clavulanic acid, sulbactam and tazobactam, but instead are inhibited by metal chelators such as EDTA and o-phenanthroline [15,16,19]. Thiol compounds such as 2-omega-phenylalkyl-3-mercaptopropionic acid [20] and N-(2mercaptoethyl)-2-phenylacetamide [21] are also competitive inhibitors. However, thus far the therapeutic potential of these inhibitors has not been demonstrated. MBLs have been found in widely distributed bacteria such as Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii [14,15,22,23]. VIM and IMP are the most frequently acquired subclasses of B enzymes [16]. MBLs show significant diversity of the active site, catalytic properties, and metal ion requirements and have been divided into three subclasses: B1, B2, and B3 [2,15,16]. Subclass B1 includes several chromosomally encoded enzymes BcII, Bacillus cereus [24], CcrA, Bacteroides fragilis [25], BlaB, Chryseobacterium meningosepticum [26], and transferable VIM, IMP, SPM, and GIM type enzymes [15,27,28]. Subclass B2 includes CphA [29] and ImiS [30] lactamases from the Aeromonas species and Sfh-I from Serratia fonticola [31]. Subclass B3 is represented by L1 from Stenotrophomonas maltophilia [32,33], FEZ-1 (...truncated)