Structural and Kinetic Studies on Metallo-β-lactamase IMP-1

Griffin, Dionne H.; Richmond, Timothy; Sanchez, Carlo; Moller, Abraham Jon; Breece, Robert M.; Tierney, David L.; Bennett, Brian; Crowder, Michael W.

doi:10.1021/bi200839h

Cited by 42 publications

(87 citation statements)

References 71 publications

(162 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…At this point it may be necessary to briefly discuss the connection between the tighter and weaker metal ion binding sites (characterised by K d1 and K d2 ) and the two available binding sites in the catalytic centre, labelled Zn1 and Zn2 (Figure 4). In B1-and B3-type MBLs Zn1 is likely to be associated with K d1 , supported, for instance, by crystallographic data that indicate a higher metal ion occupancy for this site than Zn2 [35] [36] [46] [59] [64]. An exception appears to be the B3-type MBL GOB from Elizabethkingia meningoseptica, where H116 in the Zn1 position (Figure 4) is replaced by a glutamine; a combination of kinetic and spectroscopic data indicate that this enzyme operates in mononuclear form with the metal ion bound to Zn2 (i.e.…”

Section: Interactions Between Mbls and Metal Ionsmentioning

confidence: 93%

“…Positive cooperativity for metal ion binding has also been reported for other B1-type MBLs, i.e. the enzymes CcrA [95]- [99] and IMP-1 [36] [41]- [46]. In contrast, the B1 MBL Bla2 from B. anthracis appears to bind the two Zn(II) in sequential order, leading to the speculation that this enzyme may be active in its mononuclear form under physiological conditions [100].…”

Section: Interactions Between Mbls and Metal Ionsmentioning

confidence: 93%

See 1 more Smart Citation

Metallo-β-Lactamases: A Major Threat to Human Health

Phelan¹,

Miraula²,

Selleck³

et al. 2014

AJMB

View full text Add to dashboard Cite

Antibiotic resistance is one of the most significant challenges facing global healthcare. Since the 1940s, antibiotics have been used to fight infections, initially with penicillin and subsequently with various derivatives including cephalosporins, carbapenams and monobactams. A common characteristic of these antibiotics is the four-membered β-lactam ring. Alarmingly, in recent years an increasing number of bacteria have become resistant to these antibiotics. A major strategy employed by these pathogens is to use Zn(II)-dependent enzymes, the metallo-β-lactamases (MBLs), which hydrolyse the β-lactam ring. Clinically useful MBL inhibitors are not yet available. Consequently, MBLs remain a major threat to human health. In this review biochemical properties of MBLs are discussed, focusing in particular on the interactions between the enzymes and the functionally essential metal ions. The precise role(s) of these metal ions is still debated and may differ between different MBLs. However, since they are required for catalysis, their binding site may present an alternative target for inhibitor design.

show abstract

Section: Interactions Between Mbls and Metal Ionsmentioning

confidence: 93%

Section: Interactions Between Mbls and Metal Ionsmentioning

confidence: 93%

Metallo-β-Lactamases: A Major Threat to Human Health

Phelan¹,

Miraula²,

Selleck³

et al. 2014

AJMB

View full text Add to dashboard Cite

show abstract

“…Zn(II)-bound anionic intermediates of chromogenic ␤-lactams, such as nitrocefin, have been observed during their hydrolysis catalyzed by the MBLs CcrA (4), L1 (5), NDM-1 (6), and VIM-2 (7). For imipenemase (IMP-1), there might be a nitrocefin intermediate (8), although other studies negate this (9,10). Tioni and coworkers also observed imipenem and meropenem intermediates in BcII (11).…”

mentioning

confidence: 99%

“…In IMP-1, no intermediate is seen with an [E]-to-[S] ratio of 1:1 for meropenem (Fig. 1E) or nitrocefin (9,10), suggesting that k 3 is Ͼk 2 . However, the fact that intermediate can accumulate at a higher initial [S] (Fig.…”

mentioning

confidence: 99%

Meropenem and Chromacef Intermediates Observed in IMP-25 Metallo-β-Lactamase-Catalyzed Hydrolysis

Oelschlaeger

Aitha

Yang

et al. 2015

Antimicrob Agents Chemother

Self Cite

View full text Add to dashboard Cite

Metallo-␤-lactamases (MBLs) efficiently inactivate most ␤-lactam antibacterials and have recently raised concerns due to this broad substrate spectrum, their global spread in various Gram-negative bacteria, and the absence of inhibitors for clinical use (1-3). Zn(II)-bound anionic intermediates of chromogenic ␤-lactams, such as nitrocefin, have been observed during their hydrolysis catalyzed by the MBLs CcrA (4), L1 (5), NDM-1 (6), and VIM-2 (7). For imipenemase (IMP-1), there might be a nitrocefin intermediate (8), although other studies negate this (9, 10). Tioni and coworkers also observed imipenem and meropenem intermediates in BcII (11). Recently, we found that the variant IMP-25 hydrolyzes meropenem more efficiently than IMP-1 and IMP-6 (12). IMP-6 was previously reported to confer resistance to carbapenems, especially meropenem (13). The increasing k cat for meropenem hydrolysis in the order IMP-1 ¡ IMP-6 ¡ IMP-25 (22 Ϯ 1 s Ϫ1 ¡ 60 Ϯ 10 s Ϫ1 ¡ 100 Ϯ 10 s Ϫ1 , respectively) also translates into increasing MICs of meropenem (16 g/ml ¡ 64 g/ml ¡ 128 g/ml, respectively) (12).To study in more detail the basis of the increased k cat in IMP-25 versus IMP-1, we carried out pre-steady-state kinetic experiments (6) for the hydrolysis of meropenem [6°C, 25 M enzyme (E) and substrate (S) ([E]-to-[S] ratio of 1:1) in 50 mM MOPS (morpholinepropanesulfonic acid) (pH 7.0) supplemented with 100 M Zn(II) ions]. The meropenem substrate (S) was tracked at 310 nm (due to noise at 300 nm), product (P) was tracked at 342 nm, and intermediate (I) was tracked at 390 nm. Details on the determination of extinction coefficients and conversion of the spectral data into concentrations are provided in the supplemental material. With IMP-1, a very small amount of intermediate was observed (Fig. 1E, black line), and the data could not be fitted to a two-phase association/decay function. Substrate disappeared at approximately the same exponential rate (25 Ϯ 3 s Ϫ1 ) at which product appeared (38 Ϯ 12 s Ϫ1 ) (Table 1). In contrast, with IMP-25 (Fig. 1A and B and Table 1), an initial appearance of I at 300 Ϯ 100 s Ϫ1 was followed by its disappearance at 24 Ϯ 8 s Ϫ1 . As with IMP-1, the exponential disappearance rate of S (27 Ϯ 1 s Ϫ1 ) was similar to the appearance rate of P (30 Ϯ 20 s Ϫ1 ). Thus, the observed rates of S disappearance and P appearance are indistinguishable between IMP-1 and IMP-25 under these conditions, and the uncertainty of the product formation rates was rather high. However, a clear formation and decay of intermediate was observed in IMP-25. Intermediate accumulated to no more than 6% of the initial [S], which equals [E], meaning that only up to 6% of the E molecules were bound to I.In order to explore if a clearer picture could be obtained at conditions that are more similar to steady-state conditions, we increased [S] to 100 M, 200 M, and 500 M. Several complications became apparent for the accurate quantification of S and P and, consequently, their disappearance and appearance rates, respectively, at higher concentr...

show abstract

“…The MICs of cephalothin, cefotaxime, imipenem, and meropenem correlate with 1/K m rather than with k cat /K m , consistent with the notion that the affinity between the enzyme and the substrate may determine the MIC. In the IMP-1-catalyzed conversion of the cephalosporin nitrocefin, k 2 (the rate constant for breakdown of the Michaelis complex into enzyme and product) is rate limiting (2 orders of magnitude smaller than k Ϫ1 [the rate constant for breakdown of the Michaelis complex into enzyme and substrate] [30]) and K m can be used as an approximation of K s . Indeed, K m (28 M) was observed to be comparable to K s (30 M) (Michael W. Crowder, personal communication).…”

mentioning

confidence: 99%

Biochemical Characterization of IMP-30, a Metallo-β-Lactamase with Enhanced Activity toward Ceftazidime

Pegg

Liu

LaCuran

et al. 2013

Antimicrob Agents Chemother

View full text Add to dashboard Cite

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...

show abstract

Structural and Kinetic Studies on Metallo-β-lactamase IMP-1

Cited by 42 publications

References 71 publications

Metallo-β-Lactamases: A Major Threat to Human Health

Metallo-β-Lactamases: A Major Threat to Human Health

Meropenem and Chromacef Intermediates Observed in IMP-25 Metallo-β-Lactamase-Catalyzed Hydrolysis

Biochemical Characterization of IMP-30, a Metallo-β-Lactamase with Enhanced Activity toward Ceftazidime

Contact Info

Product

Resources

About