The Sequence-Activity Relationship between Metallo-β-Lactamases IMP-1, IMP-6, and IMP-25 Suggests an Evolutionary Adaptation to Meropenem Exposure

Abstract

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 ...

Understanding the determinants of substrate specificity in IMP family metallo‐β‐lactamases: The importance of residue 262

“…15,25 However, the evolutionary advantage of expressing IMP-1-S262T versus other IMP variants will need to be defined by the clinical context in which this variant is recovered.…”

“…13 The bla IMP-1 gene with its native leader sequence for periplasmic expression was excised from the pET26b vector (kindly provided by Dr. James Spencer, University of Bristol, UK) and inserted into the pBC SK (1) vector (Stratagene, Santa Clara, CA) resulting in pBC SK(1)-bla IMP-1 . 15 To obtain pBC SK(1)-…”

“…15 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and protein quantification indicated homogeneous proteins of >95% purity and overall yields of 1.1 mg and 1.3 mg per liter culture for IMP-1-S262T and IMP-1-S262V, respectively (Table I). The percentages of protein expressed in the soluble form, determined by comparing soluble and insoluble fractions after cell lysis via SDS-PAGE, were estimated to be 70% and 50% for IMP-1-S262T and IMP-1-S262V, respectively.…”

“…14 In this study meropenem behaved like a Type II substrate, although the [5-(dimethylcarbamoyl)pryrrolidin-3-yl]sulfanyl R 2 group with a pK a of 7.4 (AstraZeneca, Mississauga, Ontario, Canada) could be neutral. 15 However, the bulkiness of the R 2 group and the axial methyl group at C 1 may account for its Type II substrate-like character. Also, it is possible that this moiety is protonated due to the microenvironment in the active site of certain MBLs, but not others.…”

“…Kinetic parameters of IMP-1 were determined previously, 15 and IMP-1-S262T and IMP-1-S262V were tested against nine antibiotics: nitrocefin, cephalothin, cefotaxime, ceftazidime, benzylpenicillin, ampicillin, imipenem, meropenem, and aztreonam. All antibiotics were hydrolyzed by these MBLs except for aztreonam.…”