The complex structures of isocitrate dehydrogenase from Clostridium thermocellum and Desulfotalea psychrophila support a new active site locking mechanism

Isocitrate dehydrogenase (IDH) catalyzes the oxidative NAD(P)+-dependent decarboxylation of isocitrate into α-ketoglutarate and CO2 and is present in organisms spanning the biological range of temperature. We have solved two crystal structures of the thermophilic Clostridium thermocellum IDH (CtIDH), a native open apo CtIDH to 2.35 Å and a quaternary complex of CtIDH with NADP+, isocitrate and Mg2+ to 2.5 Å. To compare to these a quaternary complex structure of the psychrophilic Desulfotalea psychrophila IDH (DpIDH) was also resolved to 1.93 Å. CtIDH and DpIDH showed similar global thermal stabilities with melting temperatures of 67.9 and 66.9 °C, respectively. CtIDH represents a typical thermophilic enzyme, with a large number of ionic interactions and hydrogen bonds per residue combined with stabilization of the N and C termini. CtIDH had a higher activity temperature optimum, and showed greater affinity for the substrates with an active site that was less thermolabile compared to DpIDH. The uncompensated negative surface charge and the enlarged methionine cluster in the hinge region both of which are important for cold activity in DpIDH, were absent in CtIDH. These structural comparisons revealed that prokaryotic IDHs in subfamily II have a unique locking mechanism involving Arg310, Asp251' and Arg255 (CtIDH). These interactions lock the large domain to the small domain and direct NADP+ into the correct orientation, which together are important for NADP+ selectivity.publishedVersio

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

Structural insights into TMB-1 and the role of residues 119 and 228 in substrate and inhibitor binding

Source at https://doi.org/10.1128/AAC.02602-16.Metallo-β-lactamases (MBLs) threaten the effectiveness of β-lactam antibiotics, including carbapenems, and are a concern for global public health. β-Lactam/β-lactamase inhibitor combinations active against class A and class D carbapenemases are used, but no clinically useful MBL inhibitor is currently available. Tripoli metallo-β-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

Initial characterization of an iron superoxide dismutase from Thermobifida fusca

publishedVersio

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

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

Published version, also available at http://dx.doi.org/10.1186/s12866-015-0402-zBackground: 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

Presence of acyl-homoserine lactones in 57 members of the Vibrionaceae family

Aims: The aim of this study was to use a sensitive method to screen and quantify 57 Vibrionaceae strains for the production of acyl-homoserine lactones (AHLs) and map the resulting AHL profiles onto a host phylogeny. Methods and Results: We used a high-performance liquid chromatography– tandem mass spectrometry (HPLC-MS/MS) protocol to measure AHLs in spent media after bacterial growth. First, the presence/absence of AHLs (qualitative analysis) was measured to choose internal standard for subsequent quantitative AHL measurements. We screened 57 strains from three genera (Aliivibrio, Photobacterium and Vibrio) of the same family (i.e. Vibrionaceae). Our results show that about half of the isolates produced multiple AHLs, typically at 25–5000 nmol l-1 . Conclusions: This work shows that production of AHL quorum sensing signals is found widespread among Vibrionaceae bacteria and that closely related strains typically produce similar AHL profiles. Significance and Impact of the Study: The AHL detection protocol presented in this study can be applied to a broad range of bacterial samples and may contribute to a wider mapping of AHL production in bacteria, for example, in clinically relevant strains

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

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The complex structures of isocitrate dehydrogenase from Clostridium thermocellum and Desulfotalea psychrophila support a new active site locking mechanism

Isocitrate dehydrogenase (IDH) catalyzes the oxidative NAD(P)+-dependent decarboxylation of isocitrate into α-ketoglutarate and CO2 and is present in organisms spanning the biological range of temperature. We have solved two crystal structures of the thermophilic Clostridium thermocellum IDH (CtIDH), a native open apo CtIDH to 2.35 Å and a quaternary complex of CtIDH with NADP+, isocitrate and Mg2+ to 2.5 Å. To compare to these a quaternary complex structure of the psychrophilic Desulfotalea psychrophila IDH (DpIDH) was also resolved to 1.93 Å. CtIDH and DpIDH showed similar global thermal stabilities with melting temperatures of 67.9 and 66.9 °C, respectively. CtIDH represents a typical thermophilic enzyme, with a large number of ionic interactions and hydrogen bonds per residue combined with stabilization of the N and C termini. CtIDH had a higher activity temperature optimum, and showed greater affinity for the substrates with an active site that was less thermolabile compared to DpIDH. The uncompensated negative surface charge and the enlarged methionine cluster in the hinge region both of which are important for cold activity in DpIDH, were absent in CtIDH. These structural comparisons revealed that prokaryotic IDHs in subfamily II have a unique locking mechanism involving Arg310, Asp251' and Arg255 (CtIDH). These interactions lock the large domain to the small domain and direct NADP+ into the correct orientation, which together are important for NADP+ selectivity