Structural and biochemical characterization of the environmental MBLs MYO-1, ECV-1 and SHD-1

BACKGROUND: MBLs form a large and heterogeneous group of bacterial enzymes conferring resistance to β-lactam antibiotics, including carbapenems. A large environmental reservoir of MBLs has been identified, which can act as a source for transfer into human pathogens. Therefore, structural investigation of environmental and clinically rare MBLs can give new insights into structure-activity relationships to explore the role of catalytic and second shell residues, which are under selective pressure. OBJECTIVES: To investigate the structure and activity of the environmental subclass B1 MBLs MYO-1, SHD-1 and ECV-1. METHODS: The respective genes of these MBLs were cloned into vectors and expressed in Escherichia coli. Purified enzymes were characterized with respect to their catalytic efficiency (kcat/Km). The enzymatic activities and MICs were determined for a panel of different β-lactams, including penicillins, cephalosporins and carbapenems. Thermostability was measured and structures were solved using X-ray crystallography (MYO-1 and ECV-1) or generated by homology modelling (SHD-1). RESULTS: Expression of the environmental MBLs in E. coli resulted in the characteristic MBL profile, not affecting aztreonam susceptibility and decreasing susceptibility to carbapenems, cephalosporins and penicillins. The purified enzymes showed variable catalytic activity in the order of <5% to ∼70% compared with the clinically widespread NDM-1. The thermostability of ECV-1 and SHD-1 was up to 8\ub0C higher than that of MYO-1 and NDM-1. Using solved structures and molecular modelling, we identified differences in their second shell composition, possibly responsible for their relatively low hydrolytic activity. CONCLUSIONS: These results show the importance of environmental species acting as reservoirs for MBL-encoding genes

Comparative Biochemical and Computational Study of the Role of Naturally Occurring Mutations at Ambler Positions 104 and 170 in GES β-Lactamases ▿

In GES-type β-lactamases, positions 104 and 170 are occupied by Glu or Lys and by Gly, Asn, or Ser, respectively. Previous studies have indicated an important role of these amino acids in the interaction with β-lactams, although their precise role, especially that of residue 104, remains uncertain. In this study, we constructed GES-1 (Glu104, Gly170), GES-2 (Glu104, Asn170), GES-5 (Glu104, Ser170), GES-6 (Lys104, Ser170), GES-7 (Lys104, Gly170), and GES-13 (Lys104, Asn170) by site-specific mutagenesis and compared their hydrolytic properties. Isogenic comparisons of β-lactam resistance levels conferred by these GES variants were also performed. Data indicated the following patterns: (i) Lys104-containing enzymes exhibited enhanced hydrolysis of oxyimino-cephalosporins and reduced efficiency against imipenem in relation to enzymes possessing Glu104, (ii) Asn170-containing enzymes showed reduced hydrolysis rates of penicillins and older cephalosporins, (iii) Ser170 enabled GES to hydrolyze cefoxitin efficiently, and (iv) Asn170 and Ser170 increased the carbapenemase character of GES enzymes but reduced their activity against ceftazidime. Molecular dynamic simulations of GES apoenzyme models, as well as construction of GES structures complexed with cefoxitin and an achiral ceftazidime-like boronic acid, provided insights into the catalytic behavior of the studied mutants. There were indications that an increased stability of the hydrogen bonding network of Glu166-Lys73-Ser70 and an altered positioning of Trp105 correlated with the substrate spectra, especially with acylation of GES by imipenem. Furthermore, likely effects of Ser170 on GES interactions with cefoxitin and of Lys104 on interactions with oxyimino-cephalosporins were revealed. Overall, the data unveiled the importance of residues 104 and 170 in the function of GES enzymes

Detection of carbapenemase producing enterobacteria using an ion sensitive field effect transistor sensor

Abstract The timely and accurate detection of carbapenemase-producing Enterobacterales (CPE) is imperative to manage this worldwide problem in an effective fashion. Herein we addressed the question of whether the protons produced during imipenem hydrolysis could be detected using an ion sensitive field effect transistor (ISFET). Application of the methodology on enzyme preparations showed that the sensor is able to detect carbapenemases of the NDM, IMP, KPC and NMC-A types at low nanomolar concentrations while VIM and OXA-48 responded at levels above 100 nM. Similar results were obtained when CPE cell suspensions were tested; NDM, IMP, NMC-A and KPC producers caused fast reductions of the output potential. Reduction rates with VIM-type and especially OXA-48 producing strains were significantly lower. Based on results with selected CPEs and carbapenemase-negative enterobacteria, a threshold of 10 mV drop at 30 min was set. Applying this threshold, the method exhibited 100% sensitivity for NDM, IMP and KPC and 77.3% for VIM producers. The OXA-48-positive strains failed to pass the detection threshold. A wide variety of carbapenemase-negative control strains were all classified as negative (100% specificity). In conclusion, an ISFET-based approach may have the potential to be routinely used for non OXA-48-like CPE detection in the clinical laboratory

Detection of carbapenemase producing enterobacteria using an ion sensitive field effect transistor sensor

The timely and accurate detection of carbapenemase-producing Enterobacterales (CPE) is imperative to manage this worldwide problem in an effective fashion. Herein we addressed the question of whether the protons produced during imipenem hydrolysis could be detected using an ion sensitive field effect transistor (ISFET). Application of the methodology on enzyme preparations showed that the sensor is able to detect carbapenemases of the NDM, IMP, KPC and NMC-A types at low nanomolar concentrations while VIM and OXA-48 responded at levels above 100 nM. Similar results were obtained when CPE cell suspensions were tested; NDM, IMP, NMC-A and KPC producers caused fast reductions of the output potential. Reduction rates with VIM-type and especially OXA-48 producing strains were significantly lower. Based on results with selected CPEs and carbapenemase-negative enterobacteria, a threshold of 10 mV drop at 30 min was set. Applying this threshold, the method exhibited 100% sensitivity for NDM, IMP and KPC and 77.3% for VIM producers. The OXA-48-positive strains failed to pass the detection threshold. A wide variety of carbapenemase-negative control strains were all classified as negative (100% specificity). In conclusion, an ISFET-based approach may have the potential to be routinely used for non OXA-48-like CPE detection in the clinical laboratory

Extended-Spectrum Properties of CMY-30, a Val211Gly Mutant of CMY-2 Cephalosporinase▿

CMY-30, a Val211Gly mutant of CMY-2 cephalosporinase, was derived by mutagenesis. The hydrolytic efficiency of CMY-30 against expanded-spectrum cephalosporins was higher than that of CMY-2 due to increased kcat values. Findings indicate a role of the Ω loop residue 211 in determining the substrate specificities of CMYs also corroborated by modeling studies

Targeting the YXXΦ Motifs of the SARS Coronaviruses 1 and 2 ORF3a Peptides by In Silico Analysis to Predict Novel Virus—Host Interactions

The emerging SARS-CoV and SARS-CoV-2 belong to the family of “common cold” RNA coronaviruses, and they are responsible for the 2003 epidemic and the current pandemic with over 6.3 M deaths worldwide. The ORF3a gene is conserved in both viruses and codes for the accessory protein ORF3a, with unclear functions, possibly related to viral virulence and pathogenesis. The tyrosine-based YXXΦ motif (Φ: bulky hydrophobic residue—L/I/M/V/F) was originally discovered to mediate clathrin-dependent endocytosis of membrane-spanning proteins. Many viruses employ the YXXΦ motif to achieve efficient receptor-guided internalisation in host cells, maintain the structural integrity of their capsids and enhance viral replication. Importantly, this motif has been recently identified on the ORF3a proteins of SARS-CoV and SARS-CoV-2. Given that the ORF3a aa sequence is not fully conserved between the two SARS viruses, we aimed to map in silico structural differences and putative sequence-driven alterations of regulatory elements within and adjacently to the YXXΦ motifs that could predict variations in ORF3a functions. Using robust bioinformatics tools, we investigated the presence of relevant post-translational modifications and the YXXΦ motif involvement in protein-protein interactions. Our study suggests that the predicted YXXΦ-related features may confer specific—yet to be discovered—functions to ORF3a proteins, significant to the new virus and related to enhanced propagation, host immune regulation and virulence

Structural insights into the enhanced carbapenemase efficiency of OXA-655 compared to OXA-10

Carbapenemases are the main cause of carbapenem resistance in Gram‐negative bacteria. How β‐lactamases with weak carbapenemase activity, such as the OXA‐10‐type class D β‐lactamases, contribute to anti‐bacterial drug resistance is unclear. OXA‐655 is a T26M and V117L OXA‐10 variant, recently identified from hospital wastewater. Despite exhibiting stronger carbapenemase activity towards ertapenem (ETP) and meropenem (MEM) in Escherichia coli, OXA‐655 exhibits reduced activity towards oxyimino‐substituted β‐lactams like ceftazidime. Here, we have solved crystal structures of OXA‐10 in complex with imipenem (IPM) and ETP, and OXA‐655 in complex with MEM in order to unravel the structure–function relationship and the impact of residue 117 in enzyme catalysis. The new crystal structures show that L117 is situated at a critical position with enhanced Van der Waals interactions to L155 in the omega loop. This restricts the movements of L155 and could explain the reduced ability for OXA‐655 to bind a bulky oxyimino group. The V117L replacement in OXA‐655 makes the active site S67 and the carboxylated K70 more water exposed. This could affect the supply of new deacylation water molecules required for hydrolysis and possibly the carboxylation rate of K70. But most importantly, L117 leaves more space for binding of the hydroxyethyl group in carbapenems. In summary, the crystal structures highlight the importance of residue 117 in OXA‐10 variants for carbapenemase activity. This study also illustrates the impact of a single amino acid substitution on the substrate profile of OXA‐10 and the evolutionary potential of new OXA‐10 variants

Structural insights into the enhanced carbapenemase efficiency of OXA‐655 compared to OXA‐10

Carbapenemases are the main cause of carbapenem resistance in Gram‐negative bacteria. How β‐lactamases with weak carbapenemase activity, such as the OXA‐10‐type class D β‐lactamases, contribute to anti‐bacterial drug resistance is unclear. OXA‐655 is a T26M and V117L OXA‐10 variant, recently identified from hospital wastewater. Despite exhibiting stronger carbapenemase activity towards ertapenem (ETP) and meropenem (MEM) in Escherichia coli, OXA‐655 exhibits reduced activity towards oxyimino‐substituted β‐lactams like ceftazidime. Here, we have solved crystal structures of OXA‐10 in complex with imipenem (IPM) and ETP, and OXA‐655 in complex with MEM in order to unravel the structure–function relationship and the impact of residue 117 in enzyme catalysis. The new crystal structures show that L117 is situated at a critical position with enhanced Van der Waals interactions to L155 in the omega loop. This restricts the movements of L155 and could explain the reduced ability for OXA‐655 to bind a bulky oxyimino group. The V117L replacement in OXA‐655 makes the active site S67 and the carboxylated K70 more water exposed. This could affect the supply of new deacylation water molecules required for hydrolysis and possibly the carboxylation rate of K70. But most importantly, L117 leaves more space for binding of the hydroxyethyl group in carbapenems. In summary, the crystal structures highlight the importance of residue 117 in OXA‐10 variants for carbapenemase activity. This study also illustrates the impact of a single amino acid substitution on the substrate profile of OXA‐10 and the evolutionary potential of new OXA‐10 variants