Initial characterization of an iron superoxide dismutase from Thermobifida fusca.

Superoxide dismutases (SODs) are enzymes that catalyze the dismutation of the superoxide radical anion into O2 and H2O2 in a two-step reaction. They are ubiquitous to all forms of life and four different types of metal centers are detected, dividing this class of enzymes into Cu-/Zn-, Ni-, Mn-, and Fe-SODs. In this study, a superoxide dismutase from the thermophilic bacteria Thermobifida fusca (TfSOD) was cloned and expressed before the recombinant enzyme was characterized. The enzyme was found to be active for superoxide dismutation measured by inhibition of cytochrome c oxidation and the inhibition of the autoxidation of pyrogallol. Its pH-optimum was determined to be 7.5, while it has a broad temperature optimum ranging from 20 to 90 °C. Combined with the Tm that was found to be 78.5 ± 0.5 °C at pH 8.0, TfSOD can be defined as a thermostable enzyme. Moreover, the crystal structure of TfSOD was determined and refined to 1.25 Å resolution. With electron paramagnetic resonance spectroscopy, it was confirmed that iron is the metal co-factor of TfSOD. The cell potential (Em) for the TfSOD-Fe3+/TfSOD-Fe2+ redox couple was determined to be 287 mV.

Structural insight into DNA joining: from conserved mechanisms to diverse scaffolds.

DNA ligases are diverse enzymes with essential functions in replication and repair of DNA; here we review recent advances in their structure and distribution and discuss how this contributes to understanding their biological roles and technological potential. Recent high-resolution crystal structures of DNA ligases from different organisms, including DNA-bound states and reaction intermediates, have provided considerable insight into their enzymatic mechanism and substrate interactions. All cellular organisms possess at least one DNA ligase, but many species encode multiple forms some of which are modular multifunctional enzymes. New experimental evidence for participation of DNA ligases in pathways with additional DNA modifying enzymes is defining their participation in non-redundant repair processes enabling elucidation of their biological functions. Coupled with identification of a wealth of DNA ligase sequences through genomic data, our increased appreciation of the structural diversity and phylogenetic distribution of DNA ligases has the potential to uncover new biotechnological tools and provide new treatment options for bacterial pathogens.

Side-Chain Interactions in d/l Peptide Nanotubes: Studies by Crystallography, NMR Spectroscopy and Molecular Dynamics.

Our understanding of the factors affecting the stability of cyclic d/l peptide (CP) nanotubes remains underdeveloped. In this work, we investigate the impact of side chain alignment, hydrophobicity and charge on CP nanotube stability through X-ray crystallography, NMR spectroscopy and molecular dynamics (MD) simulations. We characterise the distinct CP-CP alignments that can form and identify stable and unstable dimers by MD simulation. We measure H-bond half-lives of synthesised CPs by 1 H-D exchange experiments and find good correlation with predicted CP-CP stabilities. We find that hydrophobic amino acids improve CP dimer stability but experimentally reduce solubility. Charged amino acids either increase or decrease CP dimer stability depending on the relative orientation and composition of charged groups. X-ray crystal structures are solved for two CPs, revealing non-tubular folded conformations. Ultimately, this work will assist the educated design of stable tubular structures for potential applications in biomedicine.

Structural intermediates of a DNA-ligase complex illuminate the role of the catalytic metal ion and mechanism of phosphodiester bond formation.

DNA ligases join adjacent 5' phosphate (5'P) and 3' hydroxyl (3'OH) termini of double-stranded DNA via a three-step mechanism requiring a nucleotide cofactor and divalent metal ion. Although considerable structural detail is available for the first two steps, less is known about step 3 where the DNA-backbone is joined or about the cation role at this step. We have captured high-resolution structures of an adenosine triphosphate (ATP)-dependent DNA ligase from Prochlorococcus marinus including a Mn-bound pre-ternary ligase-DNA complex poised for phosphodiester bond formation, and a post-ternary intermediate retaining product DNA and partially occupied AMP in the active site. The pre-ternary structure unambiguously identifies the binding site of the catalytic metal ion and confirms both its role in activating the 3'OH terminus for nucleophilic attack on the 5'P group and stabilizing the pentavalent transition state. The post-ternary structure indicates that DNA distortion and most enzyme-AMP contacts remain after phosphodiester bond formation, implying loss of covalent linkage to the DNA drives release of AMP, rather than active site rearrangement. Additionally, comparisons of this cyanobacterial DNA ligase with homologs from bacteria and bacteriophage pose interesting questions about the structural origin of double-strand break joining activity and the evolution of these ATP-dependent DNA ligase enzymes.

ZN148 Is a Modular Synthetic Metallo-ß-Lactamase Inhibitor That Reverses Carbapenem Resistance in Gram-Negative Pathogens In Vivo.

Carbapenem-resistant Gram-negative pathogens are a critical public health threat and there is an urgent need for new treatments. Carbapenemases (ß-lactamases able to inactivate carbapenems) have been identified in both serine ß-lactamase (SBL) and metallo-ß-lactamase (MBL) families. The recent introduction of SBL carbapenemase inhibitors has provided alternative therapeutic options. Unfortunately, there are no approved inhibitors of MBL-mediated carbapenem-resistance and treatment options for infections caused by MBL-producing Gram-negatives are limited. Here, we present ZN148, a zinc-chelating MBL-inhibitor capable of restoring the bactericidal effect of meropenem and in vitro clinical susceptibility to carbapenems in >98% of a large international collection of MBL-producing clinical Enterobacterales strains (n = 234). Moreover, ZN148 was able to potentiate the effect of meropenem against NDM-1-producing Klebsiella pneumoniae in a murine neutropenic peritonitis model. ZN148 showed no inhibition of the human zinc-containing enzyme glyoxylase II at 500 µM, and no acute toxicity was observed in an in vivo mouse model with cumulative dosages up to 128 mg/kg. Biochemical analysis showed a time-dependent inhibition of MBLs by ZN148 and removal of zinc ions from the active site. Addition of exogenous zinc after ZN148 exposure only restored MBL activity by ∼30%, suggesting an irreversible mechanism of inhibition. Mass-spectrometry and molecular modeling indicated potential oxidation of the active site Cys221 residue. Overall, these results demonstrate the therapeutic potential of a ZN148-carbapenem combination against MBL-producing Gram-negative pathogens and that ZN148 is a highly promising MBL inhibitor that is capable of operating in a functional space not presently filled by any clinically approved compound.

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 ß-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 ß-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°C 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.

Structural studies of triazole inhibitors with promising inhibitor effects against antibiotic resistance metallo-ß-lactamases.

Metallo-ß-lactamases (MBLs) are an emerging cause of bacterial antibiotic resistance by hydrolysing all classes of ß-lactams except monobactams, and the MBLs are not inhibited by clinically available serine-ß-lactamase inhibitors. Two of the most commonly encountered MBLs in clinical isolates worldwide - the New Delhi metallo-ß-lactamase (NDM-1) and the Verona integron-encoded metallo-ß-lactamase (VIM-2) - are included in this study. A series of several NH-1,2,3-triazoles was prepared by a three-step protocol utilizing Banert cascade reaction as the key step. The inhibitor properties were evaluated in biochemical assays against the MBLs VIM-2, NDM-1 and GIM-1, and VIM-2 showed IC50 values down to nanomolar range. High-resolution crystal structures of four inhibitors in complex with VIM-2 revealed hydrogen bonds from the triazole inhibitors to Arg228 and to the backbone of Ala231 or Asn233, along with hydrophobic interactions to Trp87, Phe61 and Tyr67. The inhibitors show reduced MIC in synergy assays with Pseudomonas aeruginosa and Escherichia coli strains harbouring VIM enzymes. The obtained results will be useful for further structural guided design of MBL inhibitors.

DNA binding with a minimal scaffold: structure-function analysis of Lig E DNA ligases.

DNA ligases join breaks in the phosphodiester backbone of DNA by catalysing the formation of bonds between opposing 5'P and 3'OH ends in an adenylation-dependent manner. Catalysis is accompanied by reorientation of two core domains to provide access to the active site for cofactor utilization and enable substrate binding and product release. The general paradigm is that DNA ligases engage their DNA substrate through complete encirclement of the duplex, completed by inter-domain kissing contacts via loops or additional domains. The recent structure of a minimal Lig E-type DNA ligase, however, implies it must use a different mechanism, as it lacks any domains or loops appending the catalytic core which could complete encirclement. In the present study, we have used a structure-guided mutagenesis approach to investigate the role of conserved regions in the Lig E proteins with respect to DNA binding. We report the structure of a Lig-E type DNA ligase bound to the nicked DNA-adenylate reaction intermediate, confirming that complete encirclement is unnecessary for substrate engagement. Biochemical and biophysical measurements of point mutants to residues implicated in binding highlight the importance of basic residues in the OB domain, and inter-domain contacts to the linker.

Dissemination and Characteristics of a Novel Plasmid-Encoded Carbapenem-Hydrolyzing Class D ß-Lactamase, OXA-436, Found in Isolates from Four Patients at Six Different Hospitals in Denmark.

The diversity of OXA-48-like carbapenemases is continually expanding. In this study, we describe the dissemination and characteristics of a novel carbapenem-hydrolyzing class D ß-lactamase (CHDL) named OXA-436. In total, six OXA-436-producing Enterobacteriaceae isolates, including Enterobacter asburiae (n = 3), Citrobacter freundii (n = 2), and Klebsiella pneumoniae (n = 1), were identified in four patients in the period between September 2013 and April 2015. All three species of OXA-436-producing Enterobacteriaceae were found in one patient. The amino acid sequence of OXA-436 showed 90.4 to 92.8% identity to the amino acid sequences of other acquired OXA-48-like variants. Expression of OXA-436 in Escherichia coli and kinetic analysis of purified OXA-436 revealed an activity profile similar to that of OXA-48 and OXA-181, with activity against penicillins, including temocillin; limited or no activity against extended-spectrum cephalosporins; and activity against carbapenems. The blaOXA-436 gene was located on a conjugative ∼314-kb IncHI2/IncHI2A plasmid belonging to plasmid multilocus sequence typing sequence type 1 in a region surrounded by chromosomal genes previously identified to be adjacent to blaOXA genes in Shewanella spp. In conclusion, OXA-436 is a novel CHDL with functional properties similar to those of OXA-48-like CHDLs. The described geographical spread among different Enterobacteriaceae and the plasmid location of blaOXA-436 illustrate its potential for further dissemination.

Structural Insights into TMB-1 and the Role of Residues 119 and 228 in Substrate and Inhibitor Binding.

Metallo-ß-lactamases (MBLs) threaten the effectiveness of ß-lactam antibiotics, including carbapenems, and are a concern for global public health. ß-Lactam/ß-lactamase inhibitor combinations active against class A and class D carbapenemases are used, but no clinically useful MBL inhibitor is currently available. Tripoli metallo-ß-lactamase-1 (TMB-1) and TMB-2 are members of MBL subclass B1a, where TMB-2 is an S228P variant of TMB-1. The role of S228P was studied by comparisons of TMB-1 and TMB-2, and E119 was investigated through the construction of site-directed mutants of TMB-1, E119Q, E119S, and E119A (E119Q/S/A). All TMB variants were characterized through enzyme kinetic studies. Thermostability and crystallization analyses of TMB-1 were performed. Thiol-based inhibitors were investigated by determining the 50% inhibitory concentrations (IC50) and binding using surface plasmon resonance (SPR) for analysis of TMB-1. Thermostability measurements found TMB-1 to be stabilized by high NaCl concentrations. Steady-state enzyme kinetics analyses found substitutions of E119, in particular, substitutions associated with the penicillins, to affect hydrolysis to some extent. TMB-2 with S228P showed slightly reduced catalytic efficiency compared to TMB-1. The IC50 levels of the new thiol-based inhibitors were 0.66 µM (inhibitor 2a) and 0.62 µM (inhibitor 2b), and the equilibrium dissociation constant (KD ) of inhibitor 2a was 1.6 µM; thus, both were more potent inhibitors than l-captopril (IC50 = 47 µM; KD = 25 µM). The crystal structure of TMB-1 was resolved to 1.75 Å. Modeling of inhibitor 2b in the TMB-1 active site suggested that the presence of the W64 residue results in T-shaped π-π stacking and R224 cation-π interactions with the phenyl ring of the inhibitor. In sum, the results suggest that residues 119 and 228 affect the catalytic efficiency of TMB-1 and that inhibitors 2a and 2b are more potent inhibitors for TMB-1 than l-captopril.

Role of Residues W228 and Y233 in the Structure and Activity of Metallo-ß-Lactamase GIM-1.

Metallo-ß-lactamases (MBLs) hydrolyze virtually all ß-lactam antibiotics, including penicillins, cephalosporins, and carbapenems. The worldwide emergence of antibiotic-resistant bacteria harboring MBLs poses an increasing clinical threat. The MBL German imipenemase-1 (GIM-1) possesses an active site that is narrower and more hydrophobic than the active sites of other MBLs. The GIM-1 active-site groove is shaped by the presence of the aromatic side chains of tryptophan at residue 228 and tyrosine at residue 233, positions where other MBLs harbor hydrophilic residues. To investigate the importance of these two residues, eight site-directed mutants of GIM-1, W228R/A/Y/S and Y233N/A/I/S, were generated and characterized using enzyme kinetics, thermostability assays, and determination of the MICs of representative ß-lactams. The structures of selected mutants were obtained by X-ray crystallography, and their interactions with ß-lactam substrates were modeled in silico. Steady-state kinetics revealed that both positions are important to GIM-1 activity but that the effects of individual mutations vary depending on the ß-lactam substrate. Activity against type 1 substrates bearing electron-donating C-3/C-4 substituents (cefoxitin, meropenem) could be enhanced by mutations at position 228, whereas hydrolysis of type 2 substrates (benzylpenicillin, ampicillin, ceftazidime, imipenem) with methyl or positively charged substituents was favored by mutations at position 233. The crystal structures showed that mutations at position 228 or the Y233A variant alters the conformation of GIM-1 loop L1 rather than that of loop L3, on which the mutations are located. Taken together, these data show that point mutations at both positions 228 and 233 can influence the catalytic properties and the structure of GIM-1.

Biochemical characterization and structural analysis of a new cold-active and salt-tolerant esterase from the marine bacterium Thalassospira sp.

A gene encoding an esterase, ThaEst2349, was identified in the marine psychrophilic bacterium Thalassospira sp. GB04J01. The gene was cloned and overexpressed in E. coli as a His-tagged fusion protein. The recombinant enzyme showed optimal activity at 45 °C and the thermal stability displayed a retention of 75 % relative activity at 40 °C after 2 h. The optimal pH was 8.5 but the enzyme kept more than 75 % of its maximal activity between pH 8.0 and 9.5. ThaEst2349 also showed remarkable tolerance towards high concentrations of salt and it was active against short-chain p-nitrophenyl esters, displaying optimal activity with the acetate. The enzyme was tested for tolerance of organic solvents and the results are suggesting that it could function as an interesting candidate for biotechnological applications. The crystal structure of ThaEst2349 was determined to 1.69 Å revealing an asymmetric unit containing two chains, which also is the biological unit. The structure has a characteristic cap domain and a catalytic triad comprising Ser158, His285 and Asp255. To explain the cold-active nature of the enzyme, we compared it against thermophilic counterparts. Our hypothesis is that a high methionine content, less hydrogen bonds and less ion pairs render the enzyme more flexible at low temperatures.

Fragment-based discovery of inhibitor scaffolds targeting the metallo-ß-lactamases NDM-1 and VIM-2.

Metallo-ß-lactamases (MBLs) render bacteria resistant to ß-lactam antibiotics and are interesting drug targets to prevent the hydrolysis of ß-lactam antibiotics. So far, there are no MBL inhibitors in clinical use and particularly the design of broad spectrum inhibitors targeting several MBLs has been difficult. In this study, we report four fragments inhibiting the clinically relevant New Delhi metallo-ß-lactamase 1 (NDM-1) and Verona integron-encoded metallo-ß-lactamase 2 (VIM-2). The fragments were identified from a library using an orthogonal screening strategy combining a surface plasmon resonance (SPR) based assay and an enzyme inhibition assay. The identified fragments showed dissociation constants (KD) ranging from 181 to 2100 µM. The binding mode of the fragments was explored using QM-polarized ligand docking. All four fragments represent interesting scaffolds for the design of broad-spectrum MBL inhibitors.

Discovery of a novel covalent non-ß-lactam inhibitor of the metallo-ß-lactamase NDM-1.

The inhibition of metallo-ß-lactamases (MBL) can prevent the hydrolysis of ß-lactam antibiotics and hence is a promising strategy for the treatment of antibiotic resistant infections. In this study, we present a novel reversible covalent inhibitor of the clinically relevant MBL New Delhi metallo-ß-lactamase 1 (NDM-1). Electrospray ionization-mass spectrometry (ESI-MS) and single site directed mutagenesis were used to show that the inhibitor forms a covalent bond with Lys224 in the active site of NDM-1. The inhibitor was further characterized using an enzyme inhibition assay, a surface plasmon resonance (SPR) based biosensor assay and covalent docking. The determined inhibition constant (KI(∗)) was 580nM and the inhibition constant for the initial complex (KI) was 76µM. To our knowledge, this inhibitor is the first example for a reversible covalent non-ß-lactam inhibitor targeting NDM-1 and a promising starting point for the design of potent covalent inhibitors.

The autoinducer synthases LuxI and AinS are responsible for temperature-dependent AHL production in the fish pathogen Aliivibrio salmonicida.

BACKGROUND: Quorum sensing (QS) is a cell-to-cell communication system used by bacteria to regulate activities such as virulence, bioluminescence and biofilm formation. The most common QS signals in Gram-negative bacteria are N-acyl-homoserine lactones (AHLs). Aliivibrio salmonicida is the etiological agent of cold water vibriosis in Atlantic salmon, a disease which occurs mainly during seasons when the seawater is below 12°C. In this work we have constructed several mutants of A. salmonicida LFI1238 in order to study the LuxI/LuxR and AinS/AinR QS systems with respect to AHL production and biofilm formation. RESULTS: Using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) we found that LuxI in A. salmonicida LFI1238 is responsible for producing seven of the different AHLs, whereas AinS is responsible for producing only one. The production of these various AHLs is dependent on both cell density and growth temperature. The AHLs were efficiently produced when wild type LFI1238 was grown at 6 or 12°C, however at 16°C AHL production decreased dramatically, and LFI1238 produced less than 5% of the maximum concentrations observed at 6°C. LitR, the master regulator of QS, was found to be a positive regulator of AinS-dependent AHL production, and to a lesser extent LuxI-dependent AHL production. This implies a connection between the two systems, and both systems were found to be involved in regulation of biofilm formation. Finally, inactivation of either luxR1 or luxR2 in the lux operon significantly reduced production of LuxI-produced AHLs. CONCLUSION: LuxI and AinS are the autoinducer synthases responsible for the eight AHLs in A. salmonicida. AHL production is highly dependent on growth temperature, and a significant decrease was observed when the bacterium was grown at a temperature above its limit for disease outbreak. Numerous AHLs could offer the opportunity for fine-tuning responses to changes in the environment.

His224 alters the R2 drug binding site and Phe218 influences the catalytic efficiency of the metallo-ß-lactamase VIM-7.

Metallo-ß-lactamases (MBLs) are the causative mechanism for resistance to ß-lactams, including carbapenems, in many Gram-negative pathogenic bacteria. One important family of MBLs is the Verona integron-encoded MBLs (VIM). In this study, the importance of residues Asp120, Phe218, and His224 in the most divergent VIM variant, VIM-7, was investigated to better understand the roles of these residues in VIM enzymes through mutations, enzyme kinetics, crystal structures, thermostability, and docking experiments. The tVIM-7-D120A mutant with a tobacco etch virus (TEV) cleavage site was enzymatically inactive, and its structure showed the presence of only the Zn1 ion. The mutant was less thermostable, with a melting temperature (T(m)) of 48.5°C, compared to 55.3 °C for the wild-type tVIM-7. In the F218Y mutant, a hydrogen bonding cluster was established involving residues Asn70, Asp84, and Arg121. The tVIM-7-F218Y mutant had enhanced activity compared to wild-type tVIM-7, and a slightly higher Tm (57.1 °C) was observed, most likely due to the hydrogen bonding cluster. Furthermore, the introduction of two additional hydrogen bonds adjacent to the active site in the tVIM-7-H224Y mutant gave a higher thermostability (T(m), 62.9 °C) and increased enzymatic activity compared to those of the wild-type tVIM-7. Docking of ceftazidime in to the active site of tVIM-7, tVIM-7-H224Y, and VIM-7-F218Y revealed that the side-chain conformations of residue 224 and Arg228 in the L3 loop and Tyr67 in the L1 loop all influence possible substrate binding conformations. In conclusion, the residue composition of the L3 loop, as shown with the single H224Y mutation, is important for activity particularly toward the positively charged cephalosporins like cefepime and ceftazidime.

Synthetic cationic antimicrobial peptides bind with their hydrophobic parts to drug site II of human serum albumin.

BACKGROUND: Many biologically active compounds bind to plasma transport proteins, and this binding can be either advantageous or disadvantageous from a drug design perspective. Human serum albumin (HSA) is one of the most important transport proteins in the cardiovascular system due to its great binding capacity and high physiological concentration. HSA has a preference for accommodating neutral lipophilic and acidic drug-like ligands, but is also surprisingly able to bind positively charged peptides. Understanding of how short cationic antimicrobial peptides interact with human serum albumin is of importance for developing such compounds into the clinics. RESULTS: The binding of a selection of short synthetic cationic antimicrobial peptides (CAPs) to human albumin with binding affinities in the µM range is described. Competitive isothermal titration calorimetry (ITC) and NMR WaterLOGSY experiments mapped the binding site of the CAPs to the well-known drug site II within subdomain IIIA of HSA. Thermodynamic and structural analysis revealed that the binding is exclusively driven by interactions with the hydrophobic moieties of the peptides, and is independent of the cationic residues that are vital for antimicrobial activity. Both of the hydrophobic moieties comprising the peptides were detected to interact with drug site II by NMR saturation transfer difference (STD) group epitope mapping (GEM) and INPHARMA experiments. Molecular models of the complexes between the peptides and albumin were constructed using docking experiments, and support the binding hypothesis and confirm the overall binding affinities of the CAPs. CONCLUSIONS: The biophysical and structural characterizations of albumin-peptide complexes reported here provide detailed insight into how albumin can bind short cationic peptides. The hydrophobic elements of the peptides studied here are responsible for the main interaction with HSA. We suggest that albumin binding should be taken into careful consideration in antimicrobial peptide studies, as the systemic distribution can be significantly affected by HSA interactions.

Fluorinated captopril analogues inhibit metallo-ß-lactamases and facilitate structure determination of NDM-1 binding pose.

Bacterial resistance to the majority of clinically used ß-lactam antibiotics is a global health threat and, consequently, the driving force for the development of metallo-ß-lactamase (MBL) inhibitors. The rapid evolution of new MBLs calls for new strategies and tools for inhibitor development. In this study, we designed and developed a series of trifluoromethylated captopril analogues as probes for structural studies of enzyme-inhibitor binding. The new compounds showed activity comparable to the non-fluorinated inhibitors against the New Delhi Metallo-ß-lactamase-1 (NDM-1). The most active compound, a derivative of D-captopril, exhibited an IC50 value of 0.3 µM. Several compounds demonstrated synergistic effects, restoring the effect of meropenem and reducing the minimum inhibitory concentration (MIC) values in NDM-1 (up to 64-fold), VIM-2 (up to 8-fold) and IMP-26 (up to 8-fold) harbouring Escherichia coli. NMR spectroscopy and molecular docking of one representative inhibitor determined the binding pose in NDM-1, demonstrating that fluorinated analogues of inhibitors are a valuable tool for structural studies of MBL-inhibitor complexes.

Epistasis arises from shifting the rate-limiting step during enzyme evolution of a ß-lactamase.

Epistasis, the non-additive effect of mutations, can provide combinatorial improvements to enzyme activity that substantially exceed the gains from individual mutations. Yet the molecular mechanisms of epistasis remain elusive, undermining our ability to predict pathogen evolution and engineer biocatalysts. Here we reveal how directed evolution of a ß-lactamase yielded highly epistatic activity enhancements. Evolution selected four mutations that increase antibiotic resistance 40-fold, despite their marginal individual effects (≤2-fold). Synergistic improvements coincided with the introduction of super-stochiometric burst kinetics, indicating that epistasis is rooted in the enzyme's conformational dynamics. Our analysis reveals that epistasis stemmed from distinct effects of each mutation on the catalytic cycle. The initial mutation increased protein flexibility and accelerated substrate binding, which is rate-limiting in the wild-type enzyme. Subsequent mutations predominantly boosted the chemical steps by fine-tuning substrate interactions. Our work identifies an overlooked cause for epistasis: changing the rate-limiting step can result in substantial synergy that boosts enzyme activity.

Crystal structures of Pseudomonas aeruginosa GIM-1: active-site plasticity in metallo-ß-lactamases.

Metallo-ß-lactamases (MBLs) have rapidly disseminated worldwide among clinically important Gram-negative bacteria and have challenged the therapeutic use of ß-lactam antibiotics, particularly carbapenems. The bla(GIM-1) gene, encoding one such enzyme, was first discovered in a Pseudomonas aeruginosa isolate from 2002 and has more recently been reported in Enterobacteriaceae. Here, we present crystal structures of GIM-1 in the apo-zinc (metal-free), mono-zinc (where Cys221 was found to be oxidized), and di-zinc forms, providing nine independently refined views of the enzyme. GIM-1 is distinguished from related MBLs in possessing a narrower active-site groove defined by aromatic side chains (Trp228 and Tyr233) at positions normally occupied by hydrophilic residues in other MBLs. Our structures reveal considerable flexibility in two loops (loop 1, residues 60 to 66; loop 2, residues 223 to 242) adjacent to the active site, with open and closed conformations defined by alternative hydrogen-bonding patterns involving Trp228. We suggest that this capacity for rearrangement permits GIM-1 to hydrolyze a wide range of ß-lactams in spite of possessing a more constrained active site. Our results highlight the structural diversity within the MBL enzyme family.