Kinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems

Bottoni, Carlo; Perilli, Mariagrazia; Marcoccia, Francesca; Piccirilli, Alessandra; Pellegrini, Cristina; Colapietro, Martina; Sabatini, Alessia; Celenza, Giuseppe; Kerff, Frédéric; Amicosante, Gianfranco; Galleni, Moreno; Mercuri, Paola Sandra

doi:10.1128/aac.01703-15

Cited by 11 publications

(9 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Meanwhile, the low affinity binding of a second Zn(II) ion leads to noncompetitive enzyme inhibition. , Most of the known B2 proteins belong to the CphA family of enzymes from aquatic bacterium Aeromonas hydrophyla and other species from the genus . CphA has been characterized in detailed by Amicosante, Galleni, and co-workers. ,,,, ImiS, a related B2 enzyme from Aeromonas veronii used as a model in numerous in vitro studies by the Crowder lab, ,,, possesses a 96% identity to CphA and is classified as a CphA allele in the BLDB database (CphA11). Other members are SfhI from Serratia fonticola, the recently described PFM family, from Pseudomonas spp., and YEM-1 from Yersinia mollaretti .…”

Section: Mbls B1 B2 B3: Folding Evolution Superfamily Active Sites An...mentioning

confidence: 99%

“…This α3 helix is followed by an unusual proline-rich loop that was mutated to test its role in the substrate profile. However, individual and additive mutations of some of these Pro residues to Ala resulted in a destabilization of the protein, without broadening the substrate specificity . Instead, the combination of mutations N116H N220G was shown to greatly expand the catalytic efficiency of CphA against various cephalosporins and penicillins, albeit at the cost of reduced imipenem hydrolysis .…”

Section: Mbls B1 B2 B3: Folding Evolution Superfamily Active Sites An...mentioning

confidence: 99%

See 1 more Smart Citation

Metallo-β-lactamases in the Age of Multidrug Resistance: From Structure and Mechanism to Evolution, Dissemination, and Inhibitor Design

2021

View full text Add to dashboard Cite

Antimicrobial resistance is one of the major problems in current practical medicine. The spread of genes coding for resistance determinants among bacteria challenges the use of approved antibiotics, narrowing the options for treatment. Resistance to carbapenems, last resort antibiotics, is a major concern. Metallo-β-lactamases (MBLs) hydrolyze carbapenems, penicillins, and cephalosporins, becoming central to this problem. These enzymes diverge with respect to serine-β-lactamases by exhibiting a different fold, active site, and catalytic features. Elucidating their catalytic mechanism has been a big challenge in the field that has limited the development of useful inhibitors. This review covers exhaustively the details of the active-site chemistries, the diversity of MBL alleles, the catalytic mechanism against different substrates, and how this information has helped developing inhibitors. We also discuss here different aspects critical to understand the success of MBLs in conferring resistance: the molecular determinants of their dissemination, their cell physiology, from the biogenesis to the processing involved in the transit to the periplasm, and the uptake of the Zn(II) ions upon metal starvation conditions, such as those encountered during an infection. In this regard, the chemical, biochemical and microbiological aspects provide an integrative view of the current knowledge of MBLs.

show abstract

Section: Mbls B1 B2 B3: Folding Evolution Superfamily Active Sites An...mentioning

confidence: 99%

Section: Mbls B1 B2 B3: Folding Evolution Superfamily Active Sites An...mentioning

confidence: 99%

Metallo-β-lactamases in the Age of Multidrug Resistance: From Structure and Mechanism to Evolution, Dissemination, and Inhibitor Design

2021

View full text Add to dashboard Cite

show abstract

“…Up to date, the impact of the production of YEM-1 on the resistance pattern is unknown. Compared to the characterized sub-class B2 MBLs (14), such as CphA (15), SfhI (16) and ImiS (17), the amino acids involved in the two zinc binding sites were strictly conserved, (N116, H118 and H196 for Zn 1 and D120, C221 and H263 for Zn2). CphA is the most studied enzyme of the sub-class B2 MBLs (18–20).…”

Section: Introductionmentioning

confidence: 95%

Mutational effects on carbapenem hydrolysis of YEM-1, a new sub-class B2 metallo-β-lactamase from Yersinia mollaretii

Mercuri

Esposito

Blétard

et al. 2020

Preprint

View full text Add to dashboard Cite

1The analysis of the genome sequence of Yersinia mollaretii (Y. mollaretii) ATCC 43969 indicates 2 the presence of the bla YEM gene coding for YEM-1, a putative subclass B2 metallo-β-lactamase. The 3 objectives of our work were to produce, purify and complete the kinetic characterization of Compared to the known subclass B2 metallo−β-lactamases, YEM-1 displayed a narrowest substrate 5 profile since it is only able to hydrolyse imipenem with a high catalytic efficiency but not all the 6 other carbapenems tested such as biapenem, meropenem, doripenem and ertapenem. A possible 7 explanation of this peculiar activity profile is the presence of tyrosine 67 (loop L1), threonine 156 8 (loop L2) and serine 236 (loop L3) respectively. We showed that the substitution of Y67 broadened 9 the activity profile of the enzyme for all carbapenems but still displayed a poor activity toward the 10 other β-lactam classes. The genus Yersinia belonging to the family of Enterobacteriaceae shows three mains groups of 31 human pathogen strains, Yersinia pestis (Y. pestis), Yersinia pseudotuberculosis (Y. pseudo-32 tuberculosis) and Yersinia enterolitica (Y. enterocolitica). Y. pestis, the bacterial agent of bubonic 33 plague, escapes the macrophages action and is the cause of a lethal bacteremia without antibiotic 34 treatments (1). A new threat is the emergence of a multidrug resistant Y. pestis strain (2). Y. 35 pseudotubercolis and Y. enterocolitica are important cause of gastroenteritis in human following the 36 ingestion of undercooked or contaminated pork and vegetables (3-4). Others Yersinia species like Y. 37 fredricksenii, Y. kristensenii, Y. intermedia, Y. mollaretii, Y. bercovieri and Y. rohdei are considered 38 as not pathogen for human (5-6). Our interest was focused on Y. mollaretii. This specie, previously 39 known as Y. enterocolitica-like organism biogroup A, was renamed Yersinia mollaretii and 40 assigned in the new biogroup 3A by Wauters et al (7). The majority of those strains were isolated 41 from environmental sources such as soil, drinking water, raw vegetables, and meat but few were 42 clinical isolates from patient stools affected by gastrointestinal disease. It was demonstrated that it 43 can colonize the low level of the human ileum but the absence of human virulence markers 44 classified this strain as non-pathogenic and saprophyte (8). The sequence genome of the reference 45 strain Y. mollaretii ATCC 43969 (numbered as CCUG26331, CDC2465-87, CIP103324 and 46 DSM18520 in other biobanks) was determined (NCBI Reference Sequence: 47 NZ_AALD02000006.1). Its analysis indicated the presence of two β-lactamase genes, namely blaB 48 coding for an AmpC-like enzyme and one coding for a putative sub-class B2 metallo β-lactamase 49 called YEM-1 (YErsinia Metallo-β-lactamase -GenBank: EEQ11779.1). It is interesting to note the 50 absence of the blaA gene encoding for an Extented Spectrum class A β-Lactamase found in other 51 Yersinia species (9-12). This observation is in good agreement with the phenotypic studies showing...

show abstract

“…The active centre is located at the bottom of the β folding. Near the active pocket, there exists a long α helix (Arg140 to Leu161), which is closely followed by an unusually proline‐rich loop . To date, the crystal structure and zinc affinity of CphA have been reported by NMR and automated nano ESI‐MS; moreover, the hydrolysis mechanism of CphA to substrates also has been explored by some researches, For example, Simona et al studied the hydrolysis of biapenem catalyzed by CphA and found that Zn 2+ indirectly activated the second water molecule to facilitate the completion of the hydrolysis procedure; Wu et al studied the hydrolysis of biapenem catalyzed by CphA through QM/MM and found that the complex between enzyme and biapenem in the hydrolysis pathway was a stable intermediate or product .…”

Section: Introductionmentioning

confidence: 99%

Recognition and interaction of CphA from Aeromonas hydrophila with imipenem and biapenem by spectroscopic analysis in combination with molecular docking

Zhang

Bian

2019

J of Molecular Recognition

View full text Add to dashboard Cite

The molecular recognition and interaction of CphA from Aeromonas hydrophila with imipenem (Imip) and biapenem (Biap) were studied by means of the combined use of fluorescence spectra and molecular docking. The results showed that both the fluorescence quenching of CphA by Imip and Biap were caused through the combined dynamic and static quenching, and the latter was dominating in the process; the microenvironment and conformational of CphA were altered upon the addition of Imip and Biap from synchronous and three‐dimensional fluorescence. The binding of CphA with Imip or Biap caused a conformational change in the loop of CphA, and through the conformational change, the loop opened the binding pocket of CphA to allow for an induced fit of the newly introduced ligand. In the binding of CphA with Imip, the whole molecule entered into the active pocket of CphA. The binding was driven by enthalpy change, and the binding force between them was mainly hydrogen bonding and Van der Waals force; whereas in the binding of CphA with Biap, only the beta‐lactam ring of Biap entered into the binding pocket of CphA while the side chain was located outside the active pocket. The binding was driven by the enthalpy change and entropy change together, and the binding force between them was mainly electrostatic interaction. This study provided an insight into the recognition and binding of CphA with antibiotics, which may be helpful for designing new substrate for beta‐lactamase and developing new antibiotics resistant to superbugs.

show abstract

Kinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems

Cited by 11 publications

References 8 publications

Metallo-β-lactamases in the Age of Multidrug Resistance: From Structure and Mechanism to Evolution, Dissemination, and Inhibitor Design

Metallo-β-lactamases in the Age of Multidrug Resistance: From Structure and Mechanism to Evolution, Dissemination, and Inhibitor Design

Mutational effects on carbapenem hydrolysis of YEM-1, a new sub-class B2 metallo-β-lactamase from Yersinia mollaretii

Recognition and interaction of CphA from Aeromonas hydrophila with imipenem and biapenem by spectroscopic analysis in combination with molecular docking

Contact Info

Product

Resources

About