Carbapenemase gene blaOXA-48 detected at six freshwater sites in Northern Ireland discharging onto identified bathing locations.

Faecal contamination of surface waters has the potential to spread not only pathogenic organisms but also antimicrobial resistant organisms. During the bathing season of 2021, weekly water samples, from six selected coastal bathing locations (n = 93) and their freshwater tributaries (n = 93), in Northern Ireland (UK), were examined for concentrations of faecal indicator bacteria Escherichia coli and intestinal enterococci. Microbial source tracking involved detection of genetic markers from the genus Bacteroides using PCR assays for the general AllBac marker, the human HF8 marker and the ruminant BacR marker for the detection of human, and ruminant sources of faecal contamination. The presence of beta-lactamase genes blaOXA-48, blaKPC, and blaNDM-1 was determined using PCR assays for the investigation of antimicrobial resistance genes that are responsible for lack of efficacy in major broad-spectrum antibiotics. The beta-lactamase gene blaOXA-48 was found in freshwater tributary samples at all six locations. blaOXA-48 was detected in 83% of samples that tested positive for the human marker and 69% of samples that tested positive for the ruminant marker over all six locations. This study suggests a risk of human exposure to antimicrobial resistant bacteria where bathing waters receive at least episodically substantial transfers from such tributaries.

Genome Analysis of Enterococcus mundtii Pe103, a Human Gut-Originated Pectinolytic Bacterium.

Pectin exists in significant amounts in vegetables and fruits as a component of the plant cell wall. In human diet, pectin is not degraded by the human digestive enzymes due to its complex structure; only gut bacteria degrade pectin in the large intestine. To date, although pectin is one of the most important sources of dietary fiber in human diet, there have been only few reports on human gut-originated pectinolytic bacteria. In this study, the strain Enterococcus mundtii Pe103, a bacterium with pectin-degrading activity, was isolated from the feces of a healthy Korean adult female. Culture experiments revealed that it could grow on pectin as the sole carbon source by degrading pectin to approximately 35% within 13 h. We report the complete genome data of human gut E. mundtii Pe103. It consists of a circular chromosome (3,084,146 bps) and two plasmids (63,713 and 56,223 bps). Genomic analysis suggested that at least nine putative enzymes related to pectin degradation are present in E. mundtii Pe103. These enzymes may be involved in the degradation of pectin. The whole genome information of E. mundtii Pe103 could improve the understanding of the mechanism underlying the degradation of pectin by human gut microbiota.

Emerging Strategies to Combat ß-Lactamase Producing ESKAPE Pathogens.

Since the discovery of penicillin by Alexander Fleming in 1929 as a therapeutic agent against staphylococci, ß-lactam antibiotics (BLAs) remained the most successful antibiotic classes against the majority of bacterial strains, reaching a percentage of 65% of all medical prescriptions. Unfortunately, the emergence and diversification of ß-lactamases pose indefinite health issues, limiting the clinical effectiveness of all current BLAs. One solution is to develop ß-lactamase inhibitors (BLIs) capable of restoring the activity of ß-lactam drugs. In this review, we will briefly present the older and new BLAs classes, their mechanisms of action, and an update of the BLIs capable of restoring the activity of ß-lactam drugs against ESKAPE (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) pathogens. Subsequently, we will discuss several promising alternative approaches such as bacteriophages, antimicrobial peptides, nanoparticles, CRISPR (clustered regularly interspaced short palindromic repeats) cas technology, or vaccination developed to limit antimicrobial resistance in this endless fight against Gram-negative pathogens.

Expression and characterization of a recombinant stevioside hydrolyzing ß-glycosidase from Enterococcus casseliflavus.

The demand for steviol glycosides, non-caloric sweet components of Stevia rebaudiana Bertoni (stevia) leaves, has increased considerably as a benefit to enhance human health. However, the supply has remained challenging due to limited production, with the lack of a specific steviol glycoside hydrolyzing enzyme. In this study, a novel ß-glucosidase (EcBgl) from Enterococcus casseliflavus was cloned and expressed in Escherichia coli. An EcBgl consists of 721 amino acids corresponding to a molecular mass of 79.37 kDa. The EcBgl was purified to homogeneity, followed by enzyme characterization. The enzyme showed optimum pH and temperature at 6.0 and 37 °C, and exhibited the kinetic constants kcat/Km for pNPG and kcat/Km for stevioside of 8583 mM-1s-1 and 95.41 mM-1s-1, respectively. When compared to the stevioside hydrolyzing ß-glycosidases previously reported, EcBgl was found to be the most efficient enzyme. EcBgl also rendered hydrolysis of the stevioside to produce rubusoside, a rare steviol glycoside with a pharmaceutical solubilizing property, by cleaving at the glucose moiety. In addition, the enzyme demonstrated substantial resistance against amygdalin, so it served as a potential enzyme in agricultural and pharmaceutical applications.

Rotation mechanism of Enterococcus hirae V1-ATPase based on asymmetric crystal structures.

In various cellular membrane systems, vacuolar ATPases (V-ATPases) function as proton pumps, which are involved in many processes such as bone resorption and cancer metastasis, and these membrane proteins represent attractive drug targets for osteoporosis and cancer. The hydrophilic V(1) portion is known as a rotary motor, in which a central axis DF complex rotates inside a hexagonally arranged catalytic A(3)B(3) complex using ATP hydrolysis energy, but the molecular mechanism is not well defined owing to a lack of high-resolution structural information. We previously reported on the in vitro expression, purification and reconstitution of Enterococcus hirae V(1)-ATPase from the A(3)B(3) and DF complexes. Here we report the asymmetric structures of the nucleotide-free (2.8 Å) and nucleotide-bound (3.4 Å) A(3)B(3) complex that demonstrate conformational changes induced by nucleotide binding, suggesting a binding order in the right-handed rotational orientation in a cooperative manner. The crystal structures of the nucleotide-free (2.2 Å) and nucleotide-bound (2.7 Å) V(1)-ATPase are also reported. The more tightly packed nucleotide-binding site seems to be induced by DF binding, and ATP hydrolysis seems to be stimulated by the approach of a conserved arginine residue. To our knowledge, these asymmetric structures represent the first high-resolution view of the rotational mechanism of V(1)-ATPase.

CylA is a sequence-specific protease involved in toxin biosynthesis.

CylA is a subtilisin-like protein belonging to a recently expanded serine protease family related to class II lanthipeptide biosynthesis. As a leader peptidase, CylA is responsible for maturation of the enterococcal cytolysin, a lantibiotic important for Enterococcus faecalis virulence. In vitro reconstitution of CylA reveals that it accepts both linear and modified cytolysin peptides with a preference for cyclized peptides. Further characterization indicates that CylA activates itself by removing its N-terminal 95 amino acids. CylA achieves sequence-specific traceless cleavage of non-cognate peptides even if they are post-translationally modified, which makes the peptidase a powerful tool for mining novel lanthipeptides by providing a general strategy for leader peptide removal. Knowledge about the substrate specificity of CylA may also facilitate the development of protease inhibitors targeting cytolysin biosynthesis as a potential therapeutic approach for enterococcal infections.

Molecular modeling studies to explore the binding affinity of virtually screened inhibitor toward different aminoglycoside kinases from diverse MDR strains.

Antibiotic resistance to aminoglycoside group of antibiotics mainly occurs by aminoglycoside kinases (AKs). Thus, targeting AKs from different multidrug resistant (MDR) strains could result into inhibition of AK enzymes and ultimately the resistance. Therefore, the present study aims to target one of these AKs that is APH(3')-Ia from Acinetobacter baumannii through structure based virtual screening (SBVS) and test the binding affinity of the most stable virtually screened inhibitor with AKs from Mycobacterium tuberculosis, Acinetobacter baumannii, Enterococcus gallinarum, and Escherichia coli. SBVS investigated ZINC71575479 as a most stable inhibitor with -8.92 kcal/mol of binding energy and 0.66 µM of inhibition constant. Molecular docking results revealed that the ZINC71575479 can efficiently bind to nucleotide triphosphate (NTP) binding site of different AKs which is a known drug target site. Sequence analysis study of different AKs showed no significant similarity for active site residues; however structure superimposition study showed conserved NTP-binding domain. Molecular dynamics (MD) simulations showed stable behavior of all docked complexes with notable conformational stability of salt bridges at NTP-binding site of different AKs. Binding energy calculations revealed the interactions between key residues from NTP- binding domain of different AKs with ZINC71575479. In order to validate the MD simulations and binding energy results, crystal structure complexed with tyrphostin AG1478 a known inhibitor of AKs was kept as control. Thus, this work demonstrates the binding efficiency of ZINC71575479 toward different AKs for circumventing aminoglycoside resistance and opens avenues for the development of new antibiotics that can target diverse MDR strains with aminoglycoside resistance.

Aminoglycoside binding and catalysis specificity of aminoglycoside 2″-phosphotransferase IVa: A thermodynamic, structural and kinetic study.

BACKGROUND: Aminoglycoside O-phosphotransferases make up a large class of bacterial enzymes that is widely distributed among pathogens and confer a high resistance to several clinically used aminoglycoside antibiotics. Aminoglycoside 2″-phosphotransferase IVa, APH(2″)-IVa, is an important member of this class, but there is little information on the thermodynamics of aminoglycoside binding and on the nature of its rate-limiting step. METHODS: We used isothermal titration calorimetry, electrostatic potential calculations, molecular dynamics simulations and X-ray crystallography to study the interactions between the enzyme and different aminoglycosides. We determined the rate-limiting step of the reaction by the means of transient kinetic measurements. RESULTS: For the first time, Kd values were determined directly for APH(2″)-IVa and different aminoglycosides. The affinity of the enzyme seems to anti-correlate with the molecular weight of the ligand, suggesting a limited degree of freedom in the binding site. The main interactions are electrostatic bonds between the positively charged amino groups of aminoglycosides and Glu or Asp residues of APH. In spite of the significantly different ratio Kd/Km, there is no large difference in the transient kinetics obtained with the different aminoglycosides. We show that a product release step is rate-limiting for the overall reaction. CONCLUSIONS: APH(2″)-IVa has a higher affinity for aminoglycosides carrying an amino group in 2' and 6', but tighter bindings do not correlate with higher catalytic efficiencies. As with APH(3')-IIIa, an intermediate containing product is preponderant during the steady state. GENERAL SIGNIFICANCE: This intermediate may constitute a good target for future drug design.

Conjugal transfer of aac(6')Ie-aph(2″)Ia gene from native species and mechanism of regulation and cross resistance in Enterococcus faecalis MCC3063 by real time-PCR.

High level aminoglycoside resistance (HLAR) in the lactic acid bacteria (LAB) derived from food animals is detrimental. The aim of this study was to investigate the localization and conjugal transfer of aminoglycoside resistance genes, aac(6')Ie-aph(2″)Ia and aph(3')IIIa in different Enterococcus species. The cross resistance patterns in Enterococcus faecalis MCC3063 to clinically important aminoglycosides by real time PCR were also studied. Southern hybridization experiments revealed the presence of aac(6')Ie-aph(2″)Ia and aph(3')IIIa genes conferring HLAR in high molecular weight plasmids except in Lactobacillus plantarum. The plasmid encoded bifunctional aac(6')Ie-aph(2″)Ia gene was transferable from Enterococcus avium (n = 2), E. cecorum (n = 1), E. faecalis (n = 1) and Pediococcus lolii (n = 1) species into the recipient strain; E. faecalis JH2-2 by filter mating experiments thus indicating the possible risks of gene transfer into pathogenic strains. Molecular analysis of cross resistance patterns in native isolate of E. faecalis MCC3063 carrying aac(6')Ie-aph(2″)Ia and aph(3')IIIa gene was displayed by quantification of the mRNA levels in this study. For this, the culture was induced with increasing concentrations of gentamicin, kanamycin and streptomycin (2048, 4096, 8192, 16384 µg/mL) individually. The increasing concentrations of gentamicin and kanamycin induced the expression of the aac(6')Ie-aph(2″)Ia and aph(3')IIIa resistance genes, respectively. Interestingly, it was observed that induction with streptomycin triggered a significant fold increase in the expression of the aph(3')IIIa gene which otherwise was not known to modify the aminoglycoside. This is noteworthy as streptomycin was found to confer cross resistance to structurally unrelated kanamycin. Also, expression of the aph(3')IIIa gene when induced with streptomycin, revealed that bacteria harbouring this gene will be able to overcome streptomycin bactericidal action at specific concentrations. HLAR in E. faecalis MCC3063 may be due to the combined expression of both the aac(6')Ie-aph(2″)Ia and aph(3')IIIa genes which could be therapeutically challenging. A combined expression of both the genes in E. faecalis MCC3063 may yield HLAR which could be therapeutically challenging. The study highlights the significant alterations in the mRNA expression levels of aac(6')Ie-aph(2″)Ia and aph(3')IIIa in resistant pathogens, upon exposure to clinically vital aminoglycosides.

Biosynthesis and role of 3-methylbutanal in cheese by lactic acid bacteria: Major metabolic pathways, enzymes involved, and strategies for control.

Branched chain aldehyde, 3-methylbutanal is associated as a key flavor compound with many hard and semi-hard cheese varieties. The presence and impact of this flavor compound in bread, meat, and certain beverages has been recently documented, however its presence and consequences regarding cheese flavor were not clearly reported. This paper gives an overview of the role of 3-methylbutanal in cheese, along with the major metabolic pathways and key enzymes leading to its formation. Moreover, different strategies are highlighted for the control of this particular flavor compound in specific cheese types.

Purification and characterization of glutamate decarboxylase from Enterococcus raffinosus TCCC11660.

Glutamate decarboxylase (GAD) is the sole enzyme that synthesizes γ-aminobutyric acid through the irreversible decarboxylation of L-glutamate. In this study, the purification and characterization of an unreported GAD from a novel strain of Enterococcus raffinosus TCCC11660 were investigated. The native GAD from E. raffinosus TCCC11660 was purified 32.3-fold with a recovery rate of 8.3%, using ultrafiltration and ammonium sulfate precipitation, followed by ion-exchange and size-exclusion chromatography. The apparent molecular weight of purified GAD, as determined by SDS-PAGE and size-exclusion chromatography was 55 and 110 kDa, respectively, suggesting that GAD exists as a dimer of identical subunits in solution. In the best sodium citrate buffer, metal ions of Mo6+ and Mg2+ had positive effects, while Cu2+, Fe2+, Zn2+ and Co2+ showed significant adverse effects on enzyme activity. The optimum pH and temperature of GAD were determined to be 4.6 and 45 °C, while the K m and V max values for the sole L-glutamate substrate were 5.26 and 3.45 µmol L-1 min-1, respectively.

Efficient chemoenzymatic synthesis of uridine 5'-diphosphate N-acetylglucosamine and uridine 5'-diphosphate N-trifluoacetyl glucosamine with three recombinant enzymes.

Uridine 5'-diphosphate N-acetylglucosamine (UDP-GlcNAc) is a natural UDP-monosaccharide donor for bacterial glycosyltransferases, while uridine 5'-diphosphate N-trifluoacetyl glucosamine (UDP-GlcNTFA) is its synthetic mimic. The chemoenzymatic synthesis of UDP-GlcNAc and UDP-GlcNTFA was attempted by three recombinant enzymes. Recombinant N-acetylhexosamine 1-kinase was used to produce GlcNAc/GlcNTFA-1-phosphate from GlcNAc/GlcNTFA. N-acetylglucosamine-1-phosphate uridyltransferase from Escherichia coli K12 MG1655 was used to produce UDP-GlcNAc/GlcNTFA from GlcNAc/GlcNTFA-1-phosphate. Inorganic pyrophosphatase from E. coli K12 MG1655 was used to hydrolyze pyrophosphate to accelerate the reaction. The above enzymes were expressed in E. coli BL21 (DE3) and purified, respectively, and finally mixed in one-pot bioreactor. The effects of reaction conditions on the production of UDP-GlcNAc and UDP-GlcNTFA were characterized. To avoid the substrate inhibition effect on the production of UDP-GlcNAc and UDP-GlcNTFA, the reaction was performed with fed batch of substrate. Under the optimized conditions, high production of UDP-GlcNAc (59.51 g/L) and UDP-GlcNTFA (46.54 g/L) were achieved in this three-enzyme one-pot system. The present work is promising to develop an efficient scalable process for the supply of UDP-monosaccharide donors for oligosaccharide synthesis.

Action mechanism of 6, 6'-dihydroxythiobinupharidine from Nuphar japonicum, which showed anti-MRSA and anti-VRE activities.

BACKGROUND: Multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE), cause serious infections at clinical sites, for which the development of new drugs is necessary. We screened candidates for new antibiotics and investigated its action mechanism. METHODS: An antimicrobial compound was isolated from an extract of Nuphar japonicum. Its chemical structure was determined by NMR, MS, and optical rotation. We measured its minimum inhibitory concentration (MIC) using the microdilution method. The effects of the compound on DNA gyrase and DNA topoisomerase IV were investigated with DNA supercoiling, decatenation, and cleavage assay. RESULTS: We isolated and identified 6,6'-dihydroxythiobinupharidine as the antimicrobial compound. The MIC of this compound was 1-4 µg/mL against various MRSA and VRE strains. We also demonstrated that this compound inhibited DNA topoisomerase IV (IC50 was 10-15 µM), but not DNA gyrase in S. aureus, both of which are known to be the targets of quinolone antibiotics and necessary for DNA replication. However, this compound only exhibited slight cross-resistance to norfloxacin-resistant S. aureus, which indicated that DTBN might inhibit other targets besides topoisomerase IV. These results suggest that 6,6'-dihydroxythiobinupharidine may be a potent candidate or seed for novel antibacterial agents. CONCLUSIONS: DTBN from N. japonicum showed anti-MRSA and anti-VRE activities. DTBN might be involved in the inhibition of DNA topoisomerase IV. GENERAL SIGNIFICANCE: DTBN might be useful as a seed compound. The information on the inhibition mechanism of DTBN will be useful for the modification of DTBN towards developing novel anti-MRSA and anti-VRE drug.

In vitro biological evaluation of novel broad-spectrum isothiazolone inhibitors of bacterial type II topoisomerases.

OBJECTIVES: To evaluate the in vitro biological properties of a novel class of isothiazolone inhibitors of the bacterial type II topoisomerases. METHODS: Inhibition of DNA gyrase and topoisomerase IV activity was assessed using DNA supercoiling and decatenation assays. MIC and MBC were determined according to CLSI guidelines. Antibacterial combinations were assessed using a two-dimensional chequerboard MIC method. Spontaneous frequency of resistance was measured at various multiples of the MIC. Resistant mutants were generated by serial passage at subinhibitory concentrations of antibacterials and genetic mutations were determined through whole genome sequencing. Mammalian cytotoxicity was evaluated using the HepG2 cell line. RESULTS: Representative isothiazolone compound REDX04957 and its enantiomers (REDX05967 and REDX05990) showed broad-spectrum bactericidal activity against the ESKAPE organisms, with the exception of Enterococcus spp., as well as against a variety of other human bacterial pathogens. Compounds retained activity against quinolone-resistant strains harbouring GyrA S83L and D87G mutations (MIC ≤4 mg/L). Compounds inhibited the supercoiling activity of wild-type DNA gyrase and the decatenation function of topoisomerase IV. Frequency of resistance of REDX04957 at 4× MIC was <9.1 × 10(-9). Against a panel of recent MDR isolates, REDX05967 demonstrated activity against Acinetobacter baumannii with MIC50 and MIC90 of 16 and 64 mg/L, respectively. Compounds showed a lack of cytotoxicity against HepG2 cells at 128 mg/L. CONCLUSIONS: Isothiazolone compounds show potent activity against Gram-positive and -negative pathogens with a dual targeting mechanism-of-action and a low potential for resistance development, meriting their continued investigation as broad-spectrum antibacterial agents.

Selection of Amine-Oxidizing Dairy Lactic Acid Bacteria and Identification of the Enzyme and Gene Involved in the Decrease of Biogenic Amines.

Accumulation of biogenic amines (BAs) in cheese and other foods is a matter of public health concern. The aim of this study was to identify the enzyme activities responsible for BA degradation in lactic acid bacteria which were previously isolated from traditional Sicilian and Apulian cheeses. The selected strains would control the concentration of BAs during cheese manufacture. First, 431 isolates not showing genes encoding the decarboxylases responsible for BA formation were selected using PCR-based methods. Ninety-four out of the 431 isolates degraded BAs (2-phenylethylamine, cadaverine, histamine, putrescine, spermine, spermidine, tyramine, or tryptamine) during cultivation on chemically defined medium. As shown by random amplification of polymorphic DNA-PCR and partial sequencing of the 16S rRNA gene, 78 of the 94 strains were Lactobacillus species (Lactobacillus casei, Lb. fermentum, Lb. parabuchneri, Lb. paracasei, Lb. paraplantarum, and Lb. rhamnosus), Leuconostoc species (Leuconostoc lactis and Ln. mesenteroides), Pediococcus pentosaceus, Lactococcus lactis, Streptococcus species (Streptococcus gallolyticus and S. thermophilus), Enterococcus lactis, and Weissella paramesenteroides A multicopper oxidase-hydrolyzing BA was purified from the most active strain, Lb. paracasei subsp. paracasei CB9CT. The gene encoding the multicopper oxidase was sequenced and was also detected in other amine-degrading strains of Lb. fermentum, Lb. paraplantarum, and P. pentosaceus Lb. paracasei subsp. paracasei CB9CT and another strain (CACIO6CT) of the same species that was able to degrade all the BAs were singly used as adjunct starters for decreasing the concentration of histamine and tyramine in industrial Caciocavallo cheese. The results of this study disclose a feasible strategy for increasing the safety of traditional cheeses while maintaining their typical sensorial traits. IMPORTANCE: Because high concentrations of the potentially toxic biogenic amines may be found in traditional/typical cheeses, the safety of these food items should be improved. Lactic acid bacteria selected for the ability to degrade biogenic amines may be used during cheese making to control the concentrations of biogenic amines.

Virulence determinants and production of extracellular enzymes in Enterococcus spp. from surface water sources.

Virulence factors in Enterococcus may be indicative of potential pathogenicity. The aim of this study was to determine the relationship between the presence of clinically relevant virulence genes, in Enterococcus spp. from environmental water, and their in vitro expression. One hundred and twenty-four Enterococcus isolates (seven species), from five surface water systems in the North West Province, South Africa, were screened for the presence of asa1, cylA, esp, gelE and hyl using polymerase chain reaction. The expression of cylA, hyl and gelE was determined by phenotypic assessments. Sixty-five percent of the isolates were positive for one virulence gene and 13% for two or more. Most frequently detected genes were gelE (32%) and cylA (28%). Enterococcal surface protein was absent in all isolates screened. The presence of virulence genes was correlated with their extracellular enzyme production. The results show that a large percentage of these environmental Enterococcus spp. possess virulence factors that could be expressed in vitro. This is a cause for concern and could have implications for individuals using this water for recreational and cultural purposes. Further investigation is required into the sources of these potential pathogenic Enterococcus isolates and measures to minimize their presence in water sources.

Torque generation of Enterococcus hirae V-ATPase.

V-ATPase (V(o)V1) converts the chemical free energy of ATP into an ion-motive force across the cell membrane via mechanical rotation. This energy conversion requires proper interactions between the rotor and stator in V(o)V1 for tight coupling among chemical reaction, torque generation, and ion transport. We developed an Escherichia coli expression system for Enterococcus hirae V(o)V1 (EhV(o)V1) and established a single-molecule rotation assay to measure the torque generated. Recombinant and native EhV(o)V1 exhibited almost identical dependence of ATP hydrolysis activity on sodium ion and ATP concentrations, indicating their functional equivalence. In a single-molecule rotation assay with a low load probe at high ATP concentration, EhV(o)V1 only showed the "clear" state without apparent backward steps, whereas EhV1 showed two states, "clear" and "unclear." Furthermore, EhV(o)V1 showed slower rotation than EhV1 without the three distinct pauses separated by 120° that were observed in EhV1. When using a large probe, EhV(o)V1 showed faster rotation than EhV1, and the torque of EhV(o)V1 estimated from the continuous rotation was nearly double that of EhV1. On the other hand, stepping torque of EhV1 in the clear state was comparable with that of EhV(o)V1. These results indicate that rotor-stator interactions of the V(o) moiety and/or sodium ion transport limit the rotation driven by the V1 moiety, and the rotor-stator interactions in EhV(o)V1 are stabilized by two peripheral stalks to generate a larger torque than that of isolated EhV1. However, the torque value was substantially lower than that of other rotary ATPases, implying the low energy conversion efficiency of EhV(o)V1.

Molecular basis of ADP inhibition of vacuolar (V)-type ATPase/synthase.

Reduction of ATP hydrolysis activity of vacuolar-type ATPase/synthase (V0V1) as a result of ADP inhibition occurs as part of the normal mechanism of V0V1 of Thermus thermophilus but not V0V1 of Enterococcus hirae or eukaryotes. To investigate the molecular basis for this difference, domain-swapped chimeric V1 consisting of both T. thermophilus and E. hirae enzymes were generated, and their function was analyzed. The data showed that the interaction between the nucleotide binding and C-terminal domains of the catalytic A subunit from E. hirae V1 is central to increasing binding affinity of the chimeric V1 for phosphate, resulting in reduction of the ADP inhibition. These findings together with a comparison of the crystal structures of T. thermophilus V1 with E. hirae V1 strongly suggest that the A subunit adopts a conformation in T. thermophilus V1 different from that in E. hirae V1. This key difference results in ADP inhibition of T. thermophilus V1 by abolishing the binding affinity for phosphate during ATP hydrolysis.

Purification and characterisation of the extracellular cholesterol oxidase enzyme from Enterococcus hirae.

BACKGROUND: Recently many efforts are being carried out to reduce cholesterol in foods. Out of the 50 selected isolates that were tested using the agar well diffusion method to assess their ability to decompose cholesterol, 24 bacterial isolates were screened based on their cholesterol-decomposition ability in liquid media. RESULTS: The bacterial isolate that displayed the highest cholesterol oxidase activity was identified as Enterococcus hirae. The maximal growth and cholesterol decomposition were achieved with a 1-day incubation under static conditions at 37 °C in cholesterol basal medium adjusted to pH 7 supplemented with 1 g/l cholesterol as the substrate, no additional carbon or nitrogen sources and 0.5 % CaSO4. The cholesterol oxidase enzyme (ChoX) produced by E. hirae was extracted at an (NH4)2SO4 saturation level of 80 % and purified with 79 % yield, resulting in 2.3-fold purification. The molecular weight of (ChoX) was 60 kDa. The optimal conditions required for the maximal activity of the purified COD enzyme produced by E. hirae were 30 min, 40 °C, pH 7.8, substrate concentration of 1 g/l and 200 ppm of MgCl2. The enzyme maintained approximately 36 % and 58.5 % of its activity after 18 days of storage at 4-8 °C. Also, the enzyme loss its activity by gradual thermal treatment, but it maintained 58.5 % of its activity at 95 °C for 2 hr. CONCLUSIONS: E. hirae Mil-31 isolated from milk had a great capacity to decompose cholesterol in basal medium supplemented with cholesterol under its optimal growth conditions. Decomposition process of cholesterol by this strain results from its production of cholesterol oxidase enzyme (ChoX). The highest specific enzyme activity and highest purification fold of purified enzyme were achieved after using Sephadex G-100.

Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.

V-ATPases are rotary molecular motors that generally function as proton pumps. We recently solved the crystal structures of the V1 moiety of Enterococcus hirae V-ATPase (EhV1) and proposed a model for its rotation mechanism. Here, we characterized the rotary dynamics of EhV1 using single-molecule analysis employing a load-free probe. EhV1 rotated in a counterclockwise direction, exhibiting two distinct rotational states, namely clear and unclear, suggesting unstable interactions between the rotor and stator. The clear state was analyzed in detail to obtain kinetic parameters. The rotation rates obeyed Michaelis-Menten kinetics with a maximal rotation rate (Vmax) of 107 revolutions/s and a Michaelis constant (Km) of 154 µM at 26 °C. At all ATP concentrations tested, EhV1 showed only three pauses separated by 120°/turn, and no substeps were resolved, as was the case with Thermus thermophilus V1-ATPase (TtV1). At 10 µM ATP (<>Km), the distribution of the durations of the catalytic pause was reproduced by a consecutive reaction with two time constants of 2.6 and 0.5 ms. These kinetic parameters were similar to those of TtV1. Our results identify the common properties of rotary catalysis of V1-ATPases that are distinct from those of F1-ATPases and will further our understanding of the general mechanisms of rotary molecular motors.