Periplasmic Enzyme Research Articles

Towards Bacterial Resistance via the Membrane Strategy: Enzymatic, Biophysical and Biomimetic Studies of the Lipid cis-trans Isomerase of Pseudomonas aeruginosa.

The lipid cis-trans isomerase (Cti) is a periplasmic heme-c enzyme found in several bacteria including Pseudomonas aeruginosa, a pathogen known for causing nosocomial infections. This metalloenzyme catalyzes the cis-trans isomerization of unsaturated fatty acids in order to rapidly modulate membrane fluidity in response to stresses that impede bacterial growth. As a consequence, breakthrough in the elucidation of the mechanism of this metalloenzyme might lead to new strategies to combat bacterial antibiotic resistance. We report the first comprehensive biochemical, electrochemical and spectroscopic characterization of a Cti enzyme. This has been possible by the successful purification of Cti from P. aeruginosa (Pa-Cti) in favorable yields with enzyme activity of 0.41 µmol/min/mg when tested with palmitoleic acid. Through a synergistic approach involving enzymology, site-directed mutagenesis, Raman spectroscopy, Mössbauer spectroscopy and electrochemistry, we identified the heme coordination and redox state, pinpointing Met163 as the sixth ligand of the FeII of heme-c in Pa-Cti. Significantly, the development of an innovative assay based on liposomes demonstrated for the first time that Cti catalyzes cis-trans isomerization directly using phospholipids as substrates without the need of protein partners, answering the important question about the substrate of Cti within the bacterial membrane.

Lytic transglycosylase Slt of Pseudomonas aeruginosa as a periplasmic hub protein.

Peptidoglycan is a major constituent of the bacterial cell wall. Its integrity as a polymeric edifice is critical for bacterial survival and, as such, it is a preeminent target for antibiotics. The peptidoglycan is a dynamic crosslinked polymer that undergoes constant biosynthesis and turnover. The soluble lytic transglycosylase (Slt) of Pseudomonas aeruginosa is a periplasmic enzyme involved in this dynamic turnover. Using amber-codon-suppression methodology in live bacteria, we incorporated a fluorescent chromophore into the structure of Slt. Fluorescent microscopy shows that Slt populates the length of the periplasmic space and concentrates at the sites of septation in daughter cells. This concentration persists after separation of the cells. Amber-codon-suppression methodology was also used to incorporate a photoaffinity amino acid for the capture of partner proteins. Mass-spectrometry-based proteomics identified 12 partners for Slt in vivo. These proteomics experiments were complemented with in vitro pulldown analyses. Twenty additional partners were identified. We cloned the genes and purified to homogeneity 22 identified partners. Biophysical characterization confirmed all as bona fide Slt binders. The identities of the protein partners of Slt span disparate periplasmic protein families, inclusive of several proteins known to be present in the divisome. Notable periplasmic partners (KD < 0.5 μM) include PBPs (PBP1a, KD = 0.07 μM; PBP5 = 0.4 μM); other lytic transglycosylases (SltB2, KD = 0.09 μM; RlpA, KD = 0.4 μM); a type VI secretion system effector (Tse5, KD = 0.3 μM); and a regulatory protease for alginate biosynthesis (AlgO, KD < 0.4 μM). In light of the functional breadth of its interactome, Slt is conceptualized as a hub protein within the periplasm.

A genetic screen identifies a role for oprF in Pseudomonas aeruginosa biofilm stimulation by subinhibitory antibiotics

Biofilms are surface-associated communities of bacteria that grow in a self-produced matrix of polysaccharides, proteins, and extracellular DNA (eDNA). Sub-minimal inhibitory concentrations (sub-MIC) of antibiotics induce biofilm formation, potentially as a defensive response to antibiotic stress. However, the mechanisms behind sub-MIC antibiotic-induced biofilm formation are unclear. We show that treatment of Pseudomonas aeruginosa with multiple classes of sub-MIC antibiotics with distinct targets induces biofilm formation. Further, addition of exogenous eDNA or cell lysate failed to increase biofilm formation to the same extent as antibiotics, suggesting that the release of cellular contents by antibiotic-driven bacteriolysis is insufficient. Using a genetic screen for stimulation-deficient mutants, we identified the outer membrane porin OprF and the ECF sigma factor SigX as important. Similarly, loss of OmpA – the Escherichia coli OprF homolog – prevented sub-MIC antibiotic stimulation of E. coli biofilms. Our screen also identified the periplasmic disulfide bond-forming enzyme DsbA and a predicted cyclic-di-GMP phosphodiesterase encoded by PA2200 as essential for biofilm stimulation. The phosphodiesterase activity of PA2200 is likely controlled by a disulfide bond in its regulatory domain, and folding of OprF is influenced by disulfide bond formation, connecting the mutant phenotypes. Addition of reducing agent dithiothreitol prevented sub-MIC antibiotic biofilm stimulation. Finally, activation of a c-di-GMP-responsive promoter follows treatment with sub-MIC antibiotics in the wild-type but not an oprF mutant. Together, these results show that antibiotic-induced biofilm formation is likely driven by a signaling pathway that translates changes in periplasmic redox state into elevated biofilm formation through increases in c-di-GMP.

The Bacteroidetes Q-rule and glutaminyl cyclase activity increase the stability of extracytoplasmic proteins.

Exclusively in the Bacteroidetes phylum, most proteins exported across the inner membrane via the Sec system and released into the periplasm by type I signal peptidase have N-terminal glutamine converted to pyroglutamate. The reaction is catalyzed by the periplasmic enzyme glutaminyl cyclase (QC), which is essential for the growth of Porphyromonas gingivalis and other periodontopathogens. Apparently, pyroglutamyl formation stabilizes extracytoplasmic proteins and/or protects them from proteolytic degradation in the periplasm. Given the role of P. gingivalis as the keystone pathogen in periodontitis, P. gingivalis QC is a promising target for the development of drugs to treat and/or prevent this highly prevalent chronic inflammatory disease leading to tooth loss and associated with severe systemic diseases.

In the Alphaproteobacterium Hyphomicrobium denitrificans SoxR Serves a Sulfane Sulfur-Responsive Repressor of Sulfur Oxidation.

In organisms that use reduced sulfur compounds as alternative or additional electron donors to organic compounds, transcriptional regulation of genes for enzymes involved in sulfur oxidation is needed to adjust metabolic flux to environmental conditions. However, little is known about the sensing and response to inorganic sulfur compounds such as thiosulfate in sulfur-oxidizing bacteria. In the Alphaproteobacterium Hyphomicrobium denitrificans, one strategy is the use of the ArsR-SmtB-type transcriptional regulator SoxR. We show that this homodimeric repressor senses sulfane sulfur and that it is crucial for the expression not only of sox genes encoding the components of a truncated periplasmic thiosulfate-oxidizing enzyme system but also of several other sets of genes for enzymes of sulfur oxidation. DNA binding and transcriptional regulatory activity of SoxR are controlled by polysulfide-dependent cysteine modification. The repressor uses the formation of a sulfur bridge between two conserved cysteines as a trigger to bind and release DNA and can also form a vicinal disulfide bond to orchestrate a response to oxidizing conditions. The importance of the sulfur bridge forming cysteines was confirmed by site-directed mutagenesis, mass spectrometry, and gel shift assays. In vivo, SoxR interacts directly or indirectly with a second closely related repressor, sHdrR.

Biosynthesis of silver nanoparticles using nitrate reductase from Aspergillus terreus N4 and their potential use as a non-alcoholic disinfectant.

Green technology has been developed for the quick production of stabilized silver nanoparticles (AgNPs), with the assistance of nitrate reductase from an isolated culture of Aspergillus terreus N4. The organism's intracellular and periplasmic fractions contained nitrate reductase, with the former demonstrating the highest activity of 0.20 IU/g of mycelium. When the fungus was cultivated in a medium comprising 1.056% glucose, 1.836% peptone, 0.3386% yeast extract, and 0.025% KNO3, the greatest nitrate reductase productivity of 0.3268 IU/g was achieved. Statistical modeling via response surface methodology was used to optimize the enzyme production. The periplasmic and intracellular enzyme fractions were found to convert Ag+ to Ag0, initiating synthesis within 20min, with predominant nanoparticle sizes between 25 and 30nm. By normalizing the effects of temperature, pH, AgNO3 concentration, and mycelium age with a variable shaking period for enzyme release, the production of AgNPs with the periplasmic fraction was optimized. The synthesis of nanoparticles occurred at temperatures of 30, 40, and 50°C, with the highest yield observed at 40 and 50°C during shorter incubation periods. Similarly, the nanoparticles were synthesized at pH levels of 7.0, 8.0, and 9.0, with the greatest production observed at pH 8.0 and 9.0 at lower incubation periods. The antimicrobial activity of AgNPs was demonstrated against common foodborne pathogens, including Staphylococcus aureus and Salmonella typhimurium, indicating their potential as non-alcoholic disinfectants.

E. coli Cell Lysis Induced by Lys394 Enzyme Assisted by Magnetic Nanoparticles Exposed to Non-Heating Low-Frequency Magnetic Field.

The spreading of microbial pathogens with more and more resistance to traditional low-molecular antibiotic agents demands new approaches to antibacterial therapy. The employment of bacteriophage enzymes capable of breaking bacterial cell walls has attracted much interest within this context. The specific features of the morphology of Gram-negative bacteria prevent the effective direct usage of lytic enzymes and require assistance from additional helpers to facilitate cell lysis. The current work is devoted to the study of boosting the lysis of Escherichia coli (E. coli) JM 109 and MH 1 strains induced by Lys394 bacteriophage endolysin by means of rod-like (56 × 13 nm) magnetic nanoparticles (MNPs) activated by a non-heating low-frequency magnetic field (LF MF) with a frequency of 50 Hz and a flux density of 68.5 mT in a pulse-pause mode (1 s on and 0.3 s off). According to theoretical assumptions, the mechanism of MNP assistance is presumably based upon the disordering of the outer membrane that facilitates enzyme permeation into peptidoglycans to its substrate. It is found that the effect of the LF MF reaches an almost a twofold acceleration of the enzyme reaction, resulting in almost 80 and 70%, respectively, of lysed E. coli JM 109 and MH 1 cells in 21 min. An increase in the membrane permeability was proven by two independent experiments employing β-lactamase periplasmic enzyme leakage and Nile Red (NR) hydrophobic dye fluorescence. It is shown that the outer membrane disordering of E. coli caused by exposure to LF MF nanoparticle movement leads to almost complete (more than 80%) β-lactamase release out of the cells' periplasm to the buffer suspension. Experiments with NR (displaying fluorescence in a non-polar medium only) reveal a drastic reduction in NR fluorescence intensity, reaching a change of an order of magnitude when exposed to LF MF. The data obtained provide evidence of changes in the bacterial cell wall structure. The result shown open up the prospects of non-heating LF MF application in enhancing enzyme activity against Gram-negative pathogens.

Small Molecule Inhibition of an Exopolysaccharide Modification Enzyme is a Viable Strategy To Block Pseudomonas aeruginosa Pel Biofilm Formation.

Biosynthesis of the Pel exopolysaccharide in Pseudomonas aeruginosa requires all seven genes of the pelABCDEFG operon. The periplasmic modification enzyme PelA contains a C-terminal deacetylase domain that is necessary for Pel-dependent biofilm formation. Herein, we show that extracellular Pel is not produced by a P. aeruginosa PelA deacetylase mutant. This positions PelA deacetylase activity as an attractive target to prevent Pel-dependent biofilm formation. Using a high-throughput screen (n = 69,360), we identified 56 compounds that potentially inhibit PelA esterase activity, the first enzymatic step in the deacetylase reaction. A secondary biofilm inhibition assay identified methyl 2-(2-pyridinylmethylene) hydrazinecarbodithioate (SK-017154-O) as a specific Pel-dependent biofilm inhibitor. Structure-activity relationship studies identified the thiocarbazate as a necessary functional group and that the pyridyl ring could be replaced with a phenyl substituent (compound 1). Both SK-017154-O and compound 1 inhibit Pel-dependent biofilm formation in Bacillus cereus ATCC 10987, which has a predicted extracellular PelA deacetylase in its pel operon. Michaelis-Menten kinetics determined SK-017154-O to be a noncompetitive inhibitor of PelA, while compound 1 did not directly inhibit PelA esterase activity. Cytotoxicity assays using human lung fibroblast cells showed that compound 1 is less cytotoxic than SK-017154-O. This work provides proof of concept that biofilm exopolysaccharide modification enzymes are important for biofilm formation and can serve as useful antibiofilm targets. IMPORTANCE Present in more than 500 diverse Gram-negative and 900 Gram-positive organisms, the Pel polysaccharide is one of the most phylogenetically widespread biofilm matrix determinants found to date. Partial de-N-acetylation of this α-1,4 linked N-acetylgalactosamine polymer by the carbohydrate modification enzyme PelA is required for Pel-dependent biofilm formation in Pseudomonas aeruginosa and Bacillus cereus. Given this and our observation that extracellular Pel is not produced by a P. aeruginosa PelA deactylase mutant, we developed an enzyme-based high-throughput screen and identified methyl 2-(2-pyridinylmethylene) hydrazinecarbodithioate (SK-017154-O) and its phenyl derivative as specific Pel-dependent biofilm inhibitors. Michaelis-Menten kinetics revealed SK-017154-O is a noncompetitive inhibitor and that its noncytotoxic, phenyl derivative does not directly inhibit P. aeruginosa PelA esterase activity. We provide proof of concept that exopolysaccharide modification enzymes can be targeted with small molecule inhibitors to block Pel-dependent biofilm development in both Gram-negative and Gram-positive bacteria.

Insights into the sulfur metabolism of Chlorobaculum tepidum by label-free quantitative proteomics.

Chlorobaculum tepidum is an anaerobic green sulfur bacterium which oxidizes sulfide, elemental sulfur, and thiosulfate for photosynthetic growth. It can also oxidize sulfide to produce extracellular S0 globules, which can be further oxidized to sulfate and used as an electron donor. Here, we performed label-free quantitative proteomics on total cell lysates prepared from different metabolic states, including a sulfur production state (10h post-incubation [PI]), the beginning of sulfur consumption (20h PI), and the end of sulfur consumption (40h PI), respectively. We observed an increased abundance of the sulfide:quinone oxidoreductase (Sqr) proteins in 10h PI indicating a sulfur production state. The periplasmic thiosulfate-oxidizing Sox enzymes and the dissimilatory sulfite reductase (Dsr) subunits showed an increased abundance in 20h PI, corresponding to the sulfur-consuming state. In addition, we found that the abundance of the heterodisulfide-reductase and the sulfhydrogenase operons was influenced by electron donor availability and may be associated with sulfur metabolism. Further, we isolated and analyzed the extracellular sulfur globules in the different metabolic states to study their morphology and the sulfur cluster composition, yielding 58 previously uncharacterized proteins in purified globules. Our results show that C. tepidum regulates the cellular levels of enzymes involved in sulfur metabolism in response to the availability of reduced sulfur compounds.

Roles of methionine and cysteine residues of the Escherichia coli sensor kinase HprS in reactive chlorine species sensing.

Sensor histidine kinase HprS, an oxidative stress sensor of Escherichia coli, senses reactive oxygen species (ROS) and reactive chlorine species (RCS), and is involved in the induction of oxidatively damaged protein repair periplasmic enzymes. We reinvestigated the roles of six methionine and four cysteine residues of HprS in the response to HClO, an RCS. The results of site-directed mutagenesis revealed that methionine residues in periplasmic and cytoplasmic regions (Met225) are involved in HprS activation. Interestingly, the Cys165Ser substitution reduced HprS activity, which was recovered by an additional Glu22Cys substitution. Our results demonstrate that the position of the inner membrane cysteine residues influences the extent of HprS activation in HClO sensing.

Soluble Iron Enhances Extracellular Electron Uptake by Shewanella oneidensis MR-1.

Extracellular electron transfer (EET) is a process that microorganisms use to reduce or oxidize external insoluble electron acceptors or donors. Much of our mechanistic understanding of this process is derived from studies of transmembrane cytochrome complexes and extracellular redox shuttles that mediate outward EET to anodes and external electron acceptors. In contrast, there are knowledge gaps concerning the reverse process of inward EET from external electron donors to cells. Here, we describe a role for soluble iron (exogenous FeCl2) in enhancing EET from cathodes to the model EET bacterium Shewanella oneidensis MR-1, with fumarate serving as the intracellular electron acceptor. This iron concentration-dependent electron uptake was eradicated upon addition of an iron chelator and occurred only in the presence of fumarate reductase, confirming an electron pathway from cathodes to this periplasmic enzyme. Moreover, S. oneidensis mutants lacking specific outer membrane and periplasmic cytochromes exhibited significantly decreased current levels relative to wild-type. These results indicate that soluble iron can function as an electron carrier to the EET machinery of S. oneidensis.

Diversity of the Hydroxylamine Oxidoreductase (HAO) Gene and Its Enzyme Active Site in Agricultural Field Soils.

Nitrification is a key process in the biogeochemical nitrogen cycle and a major emission source of the greenhouse gas nitrous oxide (N2O). The periplasmic enzyme hydroxylamine oxidoreductase (HAO) is involved in the oxidation of hydroxylamine to nitric oxide in the second step of nitrification, producing N2O as a byproduct. Its three-dimensional structure demonstrates that slight differences in HAO active site residues have inhibitor effects. Therefore, a more detailed understanding of the diversity of HAO active site residues in soil microorganisms is important for the development of novel nitrification inhibitors using structure-guided drug design. However, this has not yet been examined. In the present study, we investigated hao gene diversity in beta-proteobacterial ammonia-oxidizing bacteria (β-AOB) and complete ammonia-oxidizing (comammox; Nitrospira spp.) bacteria in agricultural fields using a clone library ana-lysis. A total of 1,949 hao gene sequences revealed that hao gene diversity in β-AOB and comammox bacteria was affected by the fertilizer treatment and field type, respectively. Moreover, hao sequences showed the almost complete conservation of the six HAO active site residues in both β-AOB and comammox bacteria. The diversity of nitrifying bacteria showed similarity between hao and amoA genes. The nxrB amplicon sequence revealed the dominance of Nitrospira cluster II in tea field soils. The present study is the first to reveal hao gene diversity in agricultural soils, which will accelerate the efficient screening of HAO inhibitors and evaluations of their suppressive effects on nitrification in agricultural soils.

EstG is a novel esterase required for cell envelope integrity in Caulobacter

Proper regulation of the bacterial cell envelope is critical for cell survival. Identification and characterization of enzymes that maintain cell envelope homeostasis is crucial, as they can be targets for effective antibiotics. In this study, we have identified a novel enzyme, called EstG, whose activity protects cells from a variety of lethal assaults in the ⍺-proteobacterium Caulobacter crescentus. Despite homology to transpeptidase family cell wall enzymes and an ability to protect against cell-wall-targeting antibiotics, EstG does not demonstrate biochemical activity toward cell wall substrates. Instead, EstG is genetically connected to the periplasmic enzymes OpgH and BglX, responsible for synthesis and hydrolysis of osmoregulated periplasmic glucans (OPGs), respectively. The crystal structure of EstG revealed similarities to esterases and transesterases, and we demonstrated esterase activity of EstG invitro. Using biochemical fractionation, we identified a cyclic hexamer of glucose as a likely substrate of EstG. This molecule is the first OPG described in Caulobacter and establishes a novel class of OPGs, the regulation and modification of which are important for stress survival and adaptation to fluctuating environments. Our data indicate that EstG, BglX, and OpgH comprise a previously unknown OPG pathway in Caulobacter. Ultimately, we propose that EstG is a novel enzyme that instead of acting on the cell wall, acts on cyclic OPGs to provide resistance to a variety of cellular stresses.

Selective recovery of the intracellular enzyme using hydrodynamic cavitation: Scalable approach

Hydrodynamic cavitation technology is a promising technology for the large-scale recovery of periplasmic enzymes. In the present work, we have studied a selective, scalable, and energy-saving approach for the recovery of the alcohol dehydrogenase (ADH) enzyme from the cytoplasm of yeast cells. The hybrid strategy of hypoosmotic alkaline pretreatment (0.001 M, 8 pH, 6h) followed by the enzyme recovery using hydrodynamic cavitation (6 bar, 5 passes) increases the selective release (34.81 U/mg) up to 21.59 times when it is compared with the recovery of the enzyme using hydrodynamic cavitation without pretreatment ((1.612 U/mg, 6 bar, 55 passes). The hypoosmotic alkaline pretreatment (0.001 M, 8 pH, 9h) followed by enzyme recovery using hydrodynamic cavitation (6 bar, 5 passes) increases the specific yield (energy efficiency; U/kJ) up to 58.62 times (1793.27 U/kJ, 6 bar, 5 passes) as compared to enzyme recovery using hydrodynamic cavitation without the pre-treatment (30.59 U/kJ, 6 bar, 35 passes).

Bacterial Cell Envelope Lysis and Hemotoxicity of Peptides Previously isolated From African Catfish, Clarias gariepinus

Background: The skin mucus layer of fish is endowed with biologics including, Antimicrobial peptides (AMPs) that offer a first line of defence against pathogens. Such peptides can either inhibit bacterial growth or completely kill the bacteria and hence are regarded as a viable alternative to traditional antibiotics, in addressing the ever-increasing incidences of antimicrobial resistance. However, one of the major hurdles to AMPs use is their poor haemolytic profile. As a result, a thorough evaluation of prospective AMPs’ bacterial cell membrane disruption and hemolytic potentials in the early phases of drug discovery is critical. The current study presented cell membrane destruction as well as hemo-compatibility of antimicrobial peptides previously isolated from skin mucus of African catfish, Clarias gariepinus. Methods: A previously isolated antimicrobial peptide in the skin mucus of African catfish, C. gariepinus were profiled using 15% Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The electrical conductivity and alkaline phosphatase assays were utilised to measure bacterial cell envelope lysis activity as a classical mode of action of the antimicrobial peptides. Afterwards, fresh Rabbit blood cells were then utilised for in vitro hemolytic assay. Results: The peptides were found to be about 5 kDa molecular weight with, ability to damage the bacterial cell envelope causing significant leakage in periplasmic alkaline phosphatase enzyme and cytoplasmic electrolytes. Even at the highest peptide extract concentration of 100 μg/mL, no significant hemolysis was observed on the fresh rabbit blood cells [3.63%;P&gt;.05], signifying their safety on normal mammalian cells. Conclusion: The findings of this study pointed out that antimicrobial peptides in skin mucus of C. gariepinus are potentially safe source of antimicrobial drug leads; however, further studies are still required to search for possibly maximum dose that is safe to host cells but still effective against infecting bacteria.

Mechanism for biodegradation of sulfamethazine by Bacillus cereus H38

Degradation of sulfonamides (SAs) by microorganisms has become a focus of current research. Sulfamethazine (SMZ) is a type of SA widely used in the livestock and poultry industry. However, understanding the intermediate products, degradation pathways and mechanism of SMZ biodegradation is limited at present. In this study, a SMZ degrading bacterium Bacillus cereus H38, which can use SMZ as its only carbon source, was isolated from farmland soil. The bacterium was gram-positive with rod-shaped cells. The effects of initial SMZ concentration, pH, temperature and amount of inoculation on the biodegradation of SMZ were investigated by a single factor experiment. The results showed that the maximum degradation rate of SMZ was achieved in the environmental conditions at an initial SMZ concentration of 5 mg/L, pH of 7.0, temperature of 25 °C and inoculation amount of 5%. Under these optimum degradation conditions, strain H38 can completely degrade SMZ within 3 days. The effects of intracellular enzymes, extracellular enzymes and periplasmic enzymes on the SMZ degradation process were compared. It was found that intracellular enzymes contributed the most to the biodegradation of SMZ, and the degradation rate approached 70%. Three possible intermediates were identified by LC-MS/MS, and two degradation pathways were proposed. Whole genome sequencing results showed that the genome size of strain H38 was 5,477,631 bp, including 5599 coding sequences (CDSs), and the GC content was 35.21%. In addition, functional annotation of CDSs was performed to analyze the metabolic pathways of nitrogen and sulfur in strain H38 combining genomics and bioinformatics. This study proposes new insights into the mechanism for biodegradation of SAs and will inform future research.

Characterisation of the redox centers of ethylbenzene dehydrogenase

Ethylbenzene dehydrogenase (EbDH), the initial enzyme of anaerobic ethylbenzene degradation from the beta-proteobacterium Aromatoleumaromaticum, is a soluble periplasmic molybdenum enzyme consisting of three subunits. It contains a Mo-bis-molybdopterin guanine dinucleotide (Mo-bis-MGD) cofactor and an 4Fe–4S cluster (FS0) in the α-subunit, three 4Fe–4S clusters (FS1 to FS3) and a 3Fe–4S cluster (FS4) in the β-subunit and a heme b cofactor in the γ-subunit. Ethylbenzene is hydroxylated by a water molecule in an oxygen-independent manner at the Mo-bis-MGD cofactor, which is reduced from the MoVI to the MoIV state in two subsequent one-electron steps. The electrons are then transferred via the Fe–S clusters to the heme b cofactor. In this report, we determine the midpoint redox potentials of the Mo-bis-MGD cofactor and FS1–FS4 by EPR spectroscopy, and that of the heme b cofactor by electrochemically induced redox difference spectroscopy. We obtained relatively high values of > 250 mV both for the MoVI–MoV redox couple and the heme b cofactor, whereas FS2 is only reduced at a very low redox potential, causing magnetic coupling with the neighboring FS1 and FS3. We compare the results with the data on related enzymes and interpret their significance for the function of EbDH.Graphical abstract

Structural analysis of the boronic acid β-lactamase inhibitor vaborbactam binding to Pseudomonas aeruginosa penicillin-binding protein 3.

Antimicrobial resistance (AMR) mediated by β-lactamases is the major and leading cause of resistance to penicillins and cephalosporins among Gram-negative bacteria. β-Lactamases, periplasmic enzymes that are widely distributed in the bacterial world, protect penicillin-binding proteins (PBPs), the major cell wall synthesizing enzymes, from inactivation by β-lactam antibiotics. Developing novel PBP inhibitors with a non-β-lactam scaffold could potentially evade this resistance mechanism. Based on the structural similarities between the evolutionary related serine β-lactamases and PBPs, we investigated whether the potent β-lactamase inhibitor, vaborbactam, could also form an acyl-enzyme complex with Pseudomonas aeruginosa PBP3. We found that this cyclic boronate, vaborbactam, inhibited PBP3 (IC50 of 262 μM), and its binding to PBP3 increased the protein thermal stability by about 2°C. Crystallographic analysis of the PBP3:vaborbactam complex reveals that vaborbactam forms a covalent bond with the catalytic S294. The amide moiety of vaborbactam hydrogen bonds with N351 and the backbone oxygen of T487. The carboxyl group of vaborbactam hydrogen bonds with T487, S485, and S349. The thiophene ring and cyclic boronate ring of vaborbactam form hydrophobic interactions, including with V333 and Y503. The active site of the vaborbactam-bound PBP3 harbors the often observed ligand-induced formation of the aromatic wall and hydrophobic bridge, yet the residues involved in this wall and bridge display much higher temperature factors compared to PBP3 structures bound to high-affinity β-lactams. These insights could form the basis for developing more potent novel cyclic boronate-based PBP inhibitors to inhibit these targets and overcome β-lactamases-mediated resistance mechanisms.

The bacterial protective armor against stress: The cis-trans isomerase of unsaturated fatty acids, a cytochrome-c type enzyme

Bacteria have evolved several outstanding strategies to resist to compounds or factors that compromise their survival. The first line of defense of the cell against environmental stresses is the membrane with fatty acids as fundamental building blocks of phospholipids. In this review, we focus on a periplasmic heme enzyme that catalyzes the cis-trans isomerization of unsaturated fatty acids to trigger a decrease in the fluidity of the membrane in order to rapidly counteract the danger. We particularly detailed the occurrence of such cis-trans isomerase in Nature, the different stresses that are at the origin of the double bond isomerization, the first steps in the elucidation of the mechanism of this peculiar metalloenzyme and some aspects of its regulation.

Biological characterization of Bacillus flexus strain SSAI1 transforming highly toxic arsenite to less toxic arsenate mediated by periplasmic arsenite oxidase enzyme encoded by aioAB genes

Biological characterization of Bacillus flexus strain SSAI1 transforming highly toxic arsenite to less toxic arsenate mediated by periplasmic arsenite oxidase enzyme encoded by aioAB genes