b Metallo-␤-lactamases are important determinants of antibacterial resistance. In this study, we investigate the sequence-activity relationship between the closely related enzymes IMP-1, IMP-6, and IMP-25. While IMP-1 is the more efficient enzyme across the overall spectrum of tested ␤-lactam antibacterial agents, IMP-6 and IMP-25 seem to have evolved to specifically inactivate the newer carbapenem meropenem. Molecular modeling indicates that the G235S mutation distinguishing IMP-25 from IMP-1 and IMP-6 may affect enzyme activity via Asn233. Antibacterial resistance in combination with few new antibacterial agents being developed raises concerns for the future treatment of bacterial infections (13). ␤-Lactamases (EC 3.5.2.6.), enzymes that hydrolyze and inactivate ␤-lactam antibacterials, and in particular metallo-␤-lactamases (MBLs), present a serious challenge to public health (4). The IMP-type enzymes are among the clinically most important MBLs (7,14). These enzymes hydrolyze penicillins, cephalosporins, and carbapenems, and they are not inhibited by traditional serine ␤-lactamase inhibitors, such as clavulanic acid and sulbactam (25). They utilize two Zn(II) ions as metal cofactors to bind antibiotics and activate a water molecule for nucleophilic attack. Although they cannot hydrolyze aztreonam, they are often coproduced in bacteria that produce aztreonam-hydrolyzing extended-spectrum ␤-lactamases (3).The IMP-1 enzyme was first isolated in 1991 in Japan from Serratia marcescens (19) and has since been found in several countries worldwide, as well as in multiple microorganisms, including Pseudomonas aeruginosa, members of the family Enterobacteriaceae, and Acinetobacter spp. (7,14). IMP-6, which differs from IMP-1 by an S262G mutation, was first isolated in 1996 in Japan, also from Serratia marcescens (27), and has also been identified in P. aeruginosa and Enterobacteriaceae in Japan (28) and South Korea (20,21,29). The biochemical properties of IMP-1 and IMP-6 have been compared extensively (10,16,18,27). Interestingly, IMP-1 confers higher resistance levels to the carbapenem imipenem and several other antibiotics (10, 16), whereas IMP-6 confers higher resistance levels to the newer carbapenems meropenem (27) and doripenem (this study).IMP-25, which differs from IMP-6 by a G235S mutation and from IMP-1 by two mutations, S262G and G235S (Fig. 1A), was recently isolated from P. aeruginosa in South Korea (GenBank accession no. EU541448). In this study, we investigate the sequence-activity relationship between these closely related enzymes by determining and comparing their biochemical characteristics and abilities to confer ␤-lactam resistance to Escherichia coli cells. Furthermore, we use molecular modeling to examine the role of residue 235.Plasmids pET30a-bla IMP-6 for heterologous overexpression and pBC SK(ϩ)-bla IMP-6 for MIC assays were created by PCRbased site-directed mutagenesis using pET30a-bla IMP-1 and pBC SK(ϩ)-bla IMP-1 [obtained by subcloning bla IMP-1 with leader sequence from pET26b into pBC ...

f Antibiotic resistance in bacteria is ever changing and adapting, as once-novel ␤-lactam antibiotics are losing their efficacy, primarily due to the production of ␤-lactamases. Metallo-␤-lactamases (MBLs) efficiently inactivate a broad range of ␤-lactam antibiotics, including carbapenems, and are often coexpressed with other antibacterial resistance factors. The rapid dissemination of MBLs and lack of novel antibacterials pose an imminent threat to global health. In an effort to better counter these resistanceconferring ␤-lactamases, an investigation of their natural evolution and resulting substrate specificity was employed. In this study, we elucidated the effects of different amino acid substitutions at position 67 in IMP-type MBLs on the ability to hydrolyze and confer resistance to a range of ␤-lactam antibiotics. Wild-type ␤-lactamases IMP-1 and IMP-10 and mutants IMP-1-V67A and IMP-1-V67I were characterized biophysically and biochemically, and MICs for Escherichia coli cells expressing these enzymes were determined. We found that all variants exhibited catalytic efficiencies (k cat /K m ) equal to or higher than that of IMP-1 against all tested ␤-lactams except penicillins, against which IMP-1 and IMP-1-V67I showed the highest k cat /K m values. The substrate-specific effects of the different amino acid substitutions at position 67 are discussed in light of their side chain structures and possible interactions with the substrates. Docking calculations were employed to investigate interactions between different side chains and an inhibitor used as a ␤-lactam surrogate. The differences in binding affinities determined experimentally and computationally seem to be governed by hydrophobic interactions between residue 67 and the inhibitor and, by inference, the ␤-lactam substrates.

In Gram-negative bacteria, resistance to b-lactam antibacterials is largely due to b-lactamases and is a growing public health threat. One of the most concerning b-lactamases to evolve in bacteria are the Class B enzymes, the metallo-b-lactamases (MBLs). To date, penams and cephems resistant to hydrolysis by MBLs have not yet been found. As a result of this broad substrate specificity, a better understanding of the role of catalytically important amino acids in MBLs is necessary to design novel b-lactams and inhibitors. Two MBLs, the wild type IMP-1 with serine at position 262, and an engineered variant with valine at the same position (IMP-1-S262V), were previously found to exhibit very different substrate spectra. These findings compelled us to investigate the impact of a threonine at position 262 (IMP-1-S262T) on the substrate spectrum. Here, we explore MBL sequence-structureactivity relationships by predicting and experimentally validating the effect of the S262T substitution in IMP-1. Using site-directed mutagenesis, threonine was introduced at position 262, and the IMP-1-S262T enzyme, as well as the other two enzymes IMP-1 and IMP-1-S262V, were purified and kinetic constants were determined against a range of b-lactam antibacterials. Catalytic efficiencies (k cat /K M ) obtained with IMP-1-S262T and minimum inhibitory concentrations (MICs) observed with bacterial cells expressing the protein were intermediate or comparable to the corresponding values with IMP-1 and IMP-1-S262V, validating the role of this residue in catalysis. Our results reveal the important role of IMP residue 262 in b-lactam turnover and support this approach to predict activities of certain novel MBL variants.Keywords: metallo-b-lactamase; point mutation; antibiotic resistance; enzyme evolution; IMP-1 antibody Abbreviations: BSA, bovine serum albumin; ESI-MS, electrospray ionization mass spectrometry; IPTG, isopropyl b-D-1-thiogalactopyranoside; MBL, metallo-b-lactamase; MIC, minimum inhibitory concentration; MOPS, 3-(N-morpholino)propanesulfonic acid; PAR, 4-(2-pyridylazo)resorcinol; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

b IMP-type enzymes constitute a clinically important family of metallo-␤-lactamases that has grown dramatically in the past decade to its current 45 known members. Here, we report the biochemical characterization of IMP-30 in comparison to IMP-1, from which it deviates by a single E59K mutation. Kinetics, MIC assays, docking, and molecular dynamics simulations support a scenario in which Lys59 interacts with the ceftazidime R 1 group, resulting in increased water access and enhanced turnover and MIC of ceftazidime. The rapid evolution and diversity of ␤-lactamases pose a danger to antibacterial chemotherapy and patient survival. Class B (1) or metallo-␤-lactamases (MBLs) are zinc enzymes found in opportunistic pathogens such as Gram-negative bacteria of the Pseudomonas (2), Acinetobacter (3), and Klebsiella (4) genera. MBLs inactivate antibiotics such as penicillins, cephalosporins, and carbapenems (5), can be transferred between different pathogenic bacteria, and are insensitive to clinically used serine ␤-lactamase inhibitors (6). The B1-subclass (7) IMP-type enzymes are among the clinically most significant MBLs (5, 8), and currently, 45 variants are known (www.lahey.org/Studies/other.asp#table).Pseudomonas aeruginosa was isolated in 2004 from a patient in Moscow, Russia, with bla IMP-30 encoded on a gene cassette, together with the aadB, cmlA7, and bla OXA-21 genes (9) (GenBank accession code DQ522237). IMP-30 (GenInfo identifier 102231667) deviates from IMP-1 by a single E59K mutation. Residue 59 is located in a ␤-strand that is part of a ␤-hairpin loop covering the active site of IMP-type enzymes and is part of the binding pocket of R 1 side chains of ␤-lactams (10) (Fig. 1). Here we present the first biochemical characterization of IMP-30 and susceptibility assays with Escherichia coli cells expressing the protein. Based on comparison to IMP-1 (11), computational docking using the software program AutoDock 4.2 (12), and molecular dynamics (MD) simulations using the program AMBER 10 (13), we suggest a possible role of Lys59.The pET30a-bla IMP-30 plasmid for overexpression and the pBC SK(ϩ)-bla IMP-30 plasmid for MIC assays were generated using the forward primer 5=-CAT ACT TCG TTT AAA GAA GTT AAC GGG-3= and the reverse primer 5=-CCC GTT AAC TTC TTT AAA CGA AGT ATG-3= (bold letters indicate the codon for lysine; the underlined letter indicates the changed nucleotide) via PCR-based site-directed mutagenesis from the pET30a-bla IMP-1 and pBC SK(ϩ)-bla IMP-1 plasmids (11). IMP-30 was overexpressed in the cytoplasm of E. coli C43 cells, purified, and biophysically characterized as described previously (14). SDS-PAGE analysis (15) revealed that the enzyme was purified to Ͼ95% homogeneity. The 4-(2-pyridylazo)resorcinol (PAR) assay (16) demonstrated that IMP-30 bound two (1.9 Ϯ 0.1) Zn(II) ions, and superimposable circular dichroism scans indicate a secondary structure comparable to that of IMP-1 (11, 14). The molecular mass determined by electron spray ionization mass spectrometry (QSTAR-XL tandem mass spectrome...