IMP-1 metallo-β-lactamase: effect of chelators and assessment of metal requirement by electrospray mass spectrometry

Siemann, Stefan; Brewer, Dyanne; Clarke, Anthony J.; Dmitrienko, Gary I.; Lajoie, Gilles; Viswanatha, T.

doi:10.1016/s0304-4165(02)00258-1

Cited by 60 publications

(55 citation statements)

References 49 publications

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“…3B, lanes 3,4), consistent with our previous results showing that cleavage was resistant to 4 mM EDTA and modestly inhibited by 8 mM (Hirose and Manley 1997). Strikingly, the zinc-specific chelators TPEN (N,N,NЈ,NЈ-tetrakis(2-pyridylmethyl)ethylenediamine) and OP (orthophenanthroline; Siemann et al 2002) each partially inhibited cleavage at 100 µM, far below the EDTA concentrations required for inhibition. Figure 3C extends these results, showing that inhibition by TPEN was concentration dependent and ∼50-fold more effective than EDTA.…”

Section: Cpsf-73 As the Mrna 3 Endonucleasesupporting

confidence: 89%

Evidence that polyadenylation factor CPSF-73 is the mRNA 3′ processing endonuclease

Ryan¹,

Calvo²,

Manley³

2004

RNA

162

170

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Generation of the polyadenylated 3 end of an mRNA requires an endonucleolytic cleavage followed by synthesis of the poly(A) tail. Despite the seeming simplicity of the reaction, more than a dozen polypeptides are required, and nearly all appear to be necessary for the cleavage reaction. Because of this complexity, the identity of the endonuclease has remained a mystery. Here we present evidence that a component of the cleavage-polyadenylation specificity factor CPSF-73 is the long-sought endonuclease. We first show, using site-specific labeling and UV-cross-linking, that a protein with properties of CPSF-73 is one of only two polypeptides in HeLa nuclear extract to contact the cleavage site in an AAUAAA-dependent manner. The recent identification of CPSF-73 as a possible member of the metallo-␤-lactamase family of Zn 2+ -dependent hydrolytic enzymes suggests that this contact may identify CPSF-73 as the nuclease. Supporting the significance of the putative hydrolytic lactamase domain in CPSF-73, we show that mutation of key residues predicted to be required for activity in the yeast CPSF-73 homolog result in lethality. Furthermore, in contrast to long held belief, but consistent with properties of metallo-␤-lactamases, we show that 3 cleavage is metal-dependent, likely reflecting a requirement for tightly protein-bound Zn 2+. Taken together, the available data provide strong evidence that CPSF-73 is the 3 processing endonuclease.

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Section: Cpsf-73 As the Mrna 3 Endonucleasesupporting

confidence: 89%

Evidence that polyadenylation factor CPSF-73 is the mRNA 3′ processing endonuclease

Ryan¹,

Calvo²,

Manley³

2004

RNA

162

170

View full text Add to dashboard Cite

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“…For DDPT, Lee et al (11) reduced the EDTA content of a disk from 1,900 to 760 g and added 2 mg of SMA; however, in the present study, the IPM DDPT and IPM DPT with these inhibitors showed sensitivities of only 90.9 and 43.2%, respectively (Table 1, method E; Table 2, method D). It has previously been reported that DPA had a stronger inhibitory activity on IMP-1 among the inhibitors tested (18) and that it had much less toxicity to P. aeruginosa (6). The high stability of the DPA disks, as is EDTA disks, is another advantage; our laboratory-prepared disks stored at room temperature for 5 years showed potentiation zone sizes similar to those of freshly prepared disks (unpublished data).…”

Section: Discussionsupporting

confidence: 52%

Evaluation of Double-Disk Potentiation and Disk Potentiation Tests Using Dipicolinic Acid for Detection of Metallo-β-Lactamase-Producing Pseudomonas spp. and Acinetobacter spp

Yong

Lee

Jeong³

et al. 2012

J Clin Microbiol

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bAccurate detection of metallo-␤-lactamase (MBL)-producing Pseudomonas spp. and Acinetobacter spp. became very important with the increasing prevalence of carbapenem-nonsusceptible clinical isolates. The performance of phenotypic MBL detection methods may depend on the types of MBL and the characteristics of the isolates. A high false-positive rate is a problem with EDTA-based MBL detection methods. We evaluated the performance of double-disk potentiation tests (DDPTs) and disk potentiation tests (DPTs) with dipicolinic acid (DPA) using 44 isolates of Pseudomonas spp. and Acinetobacter spp. producing IMP-1-like, VIM-2-like, and SIM-1 type MBLs. Also, we characterized P. aeruginosa isolates with positive imipenem (IPM)-DPA DDPT, but negative meropenem (MEM)-DPA DDPT, and determined possibility of improving a DDPT by using MacConkey agar. Among five different DDPT methods, the IPM-DPA 250-g method showed the highest sensitivity (97.7%) and specificity (100%). Among four DPT tests, the highest sensitivity (100%) was shown by the IPM-EDTA 1,900-g disk method, but the specificity was very low (11.4%). Five of six P. aeruginosa isolates with false-negative DDPTs with MEM-DPA 250-g disks carried bla IMP-6, and the high level resistance to MEM (MIC > 512 g/ml) was reduced by the presence of phenylalanine arginine ␤-naphtylamide. Improvement of DDPTs was observed when MacConkey agar was used instead of Mueller-Hinton agar. In conclusion, DPA is a better MBL inhibitor than EDTA for detection of Pseudomonas spp. and Acinetobacter spp. with IMP-1-like, VIM-2-like, and SIM-1-type MBLs. In DPA DDPTs, IPM disks perform better than MEM disks when the isolates are highly resistant to MEM due to the overexpression of efflux pumps. C arbapenems are often used as "antibiotics of last resort" when patients with infections become gravely ill or are suspected of harboring resistant bacteria (14). However, carbapenem-resistant Gram-negative bacilli have been increasingly reported worldwide. In Korea, carbapenem-resistant isolates of Enterobacteriaceae remains very rare, but during the period of 2001 to 2009, imipenem (IPM)-resistant Pseudomonas aeruginosa and Acinetobacter spp. increased from 17 to 26% and from 5 to 51%, respectively (10). A significant proportion of the resistance in P. aeruginosa is due to the production of VIM-2-like and IMP-1-like metallo-␤-lactamase (MBL), whereas in Acinetobacter spp. the majority are due to the production of OXA-type carbapenemase, although VIM-2-like, IMP-1-like, and SIM-1 enzyme-producing isolates have also been detected (8).Various inhibitor-based methods are commonly used to detect MBLs, but these techniques are not highly sensitive or highly specific. A disk potentiation test (DPT) is also called a combined disk test. A DPT using imipenem (IPM) and EDTA (IPM-EDTA DPT) showed a high false-positive rate with Acinetobacter spp. due to growth inhibition by EDTA alone (20). A double-disk potentiation test (DDPT) is also known as a double-disk synergy test. To reduce false-positive results from t...

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“…Another interesting finding was the apparent increase in wildtype enzyme activity, as reflected by the antibiotic MIC, with increasing concentrations of environmental zinc. Previous reports on IMP-1 have shown that protein expressed and purified from E. coli grown under rich-medium conditions contains two zinc ions per enzyme molecule and exhibits optimal catalytic function (6,18,20,53). Our current findings from defined minimal medium suggest that in vivo enzyme function is highly dependent on the availability of exogenous zinc in the growth environment of E. coli.…”

Section: Saturation Mutagenesis Of Position 221 and Identification Ofsupporting

confidence: 53%

Mutagenesis of Zinc Ligand Residue Cys221 Reveals Plasticity in the IMP-1 Metallo-β-Lactamase Active Site

Banks

Shanker

Mikulski

et al. 2012

Antimicrob Agents Chemother

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e Metallo-␤-lactamases catalyze the hydrolysis of a broad range of ␤-lactam antibiotics and are a concern for the spread of drug resistance. To analyze the determinants of enzyme structure and function, the sequence requirements for the subclass B1 IMP-1 ␤-lactamase zinc binding residue Cys221 were tested by saturation mutagenesis and evaluated for protein expression, as well as hydrolysis of ␤-lactam substrates. The results indicated that most substitutions at position 221 destabilized the enzyme. Only the enzymes containing C221D and C221G substitutions were expressed well in Escherichia coli and exhibited catalytic activity toward ␤-lactam antibiotics. Despite the lack of a metal-chelating group at position 221, the C221G enzyme exhibited high levels of catalytic activity in the presence of exogenous zinc. Molecular modeling suggests the glycine substitution is unique among substitutions in that the complete removal of the cysteine side chain allows space for a water molecule to replace the thiol and coordinate zinc at the Zn2 zinc binding site to restore function. Multiple methods were used to estimate the C221G Zn2 binding constant to be 17 to 43 M. Studies of enzyme function in vivo in E. coli grown on minimal medium showed that both IMP-1 and the C221G mutant exhibited compromised activity when zinc availability was low. Finally, substitutions at residue 121, which is the IMP-1 equivalent of the subclass B3 zinc-chelating position, failed to rescue C221G function, suggesting the coordination schemes of subclasses B1 and B3 are not interchangeable.A n increasing prevalence of antibiotic-resistant strains is reducing the available options for treating bacterial infections. ␤-Lactam antibiotics, such as the penicillins and cephalosporins, are among the most often used antimicrobial agents (31). The major contributors to ␤-lactam antibiotic resistance are ␤-lactamase enzymes, which act by hydrolyzing the four-member ␤-lactam ring (15,62). Mechanistically, this is accomplished either via an active-site serine in the class A, C, and D enzymes or through the use of one or two Zn 2ϩ ions (class B) (4). Class B metallo-␤-lactamases (M␤Ls) are a group of structurally similar enzymes that exhibit a characteristic ␣␤/␤␣ sandwich fold, with the active site located at the interface between domains. This scaffold supports up to 6 residues at the active site that coordinate either one or two zinc ions that are central to the catalytic mechanism (1,6,35). M␤Ls have the capacity to hydrolyze most clinically available ␤-lactam drugs, including extended-spectrum cephalosporins and carbapenems (8,10,32,40,59,61).The IMP-1 metallo-␤-lactamase has been identified in several nosocomial, Gram-negative, pathogenic bacteria, including Pseudomonas aeruginosa and Serratia marcesens (22, 28). The bla IMP-1 gene is carried on an integron element that facilitates genetic transfer and is likely why IMP-1 is found among multiple bacterial species (28, 58). The combination of a broad ␤-lactam antibiotic substrate profile and the genetic ca...

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IMP-1 metallo-β-lactamase: effect of chelators and assessment of metal requirement by electrospray mass spectrometry

Cited by 60 publications

References 49 publications

Evidence that polyadenylation factor CPSF-73 is the mRNA 3′ processing endonuclease

Evidence that polyadenylation factor CPSF-73 is the mRNA 3′ processing endonuclease

Evaluation of Double-Disk Potentiation and Disk Potentiation Tests Using Dipicolinic Acid for Detection of Metallo-β-Lactamase-Producing Pseudomonas spp. and Acinetobacter spp

Mutagenesis of Zinc Ligand Residue Cys221 Reveals Plasticity in the IMP-1 Metallo-β-Lactamase Active Site

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