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1.
Daniil M. Prigozhin Daniela Mavrici John P. Huizar Hilary J. Vansell Tom Alber 《The Journal of biological chemistry》2013,288(44):31549-31555
Peptidoglycan hydrolases are key enzymes in bacterial cell wall homeostasis. Understanding the substrate specificity and biochemical activity of peptidoglycan hydrolases in Mycobacterium tuberculosis is of special interest as it can aid in the development of new cell wall targeting therapeutics. In this study, we report biochemical and structural characterization of the mycobacterial N-acetylmuramyl-l-alanine amidase, Rv3717. The crystal structure of Rv3717 in complex with a dipeptide product shows that, compared with previously characterized peptidoglycan amidases, the enzyme contains an extra disulfide-bonded β-hairpin adjacent to the active site. The structure of two intermediates in assembly reveal that Zn2+ binding rearranges active site residues, and disulfide formation promotes folding of the β-hairpin. Although Zn2+ is required for hydrolysis of muramyl dipeptide, disulfide oxidation is not required for activity on this substrate. The orientation of the product in the active site suggests a role for a conserved glutamate (Glu-200) in catalysis; mutation of this residue abolishes activity. The product binds at the head of a closed tunnel, and the enzyme showed no activity on polymerized peptidoglycan. These results point to a potential role for Rv3717 in peptidoglycan fragment recycling. 相似文献
2.
Gilmore ME Bandyopadhyay D Dean AM Linnstaedt SD Popham DL 《Journal of bacteriology》2004,186(1):80-89
Bacterial spore heat resistance is primarily dependent upon dehydration of the spore cytoplasm, a state that is maintained by the spore peptidoglycan wall, the spore cortex. A peptidoglycan structural modification found uniquely in spores is the formation of muramic delta-lactam. Production of muramic delta-lactam in Bacillus subtilis requires removal of a peptide side chain from the N-acetylmuramic acid residue by a cwlD-encoded muramoyl-L-Alanine amidase. Expression of cwlD takes place in both the mother cell and forespore compartments of sporulating cells, though expression is expected to be required only in the mother cell, from which cortex synthesis derives. Expression of cwlD in the forespore is in a bicistronic message with the upstream gene ybaK. We show that ybaK plays no apparent role in spore peptidoglycan synthesis and that expression of cwlD in the forespore plays no significant role in spore peptidoglycan formation. Peptide cleavage by CwlD is apparently followed by deacetylation of muramic acid and lactam ring formation. The product of pdaA (yfjS), which encodes a putative deacetylase, has recently been shown to also be required for muramic delta-lactam formation. Expression of CwlD in Escherichia coli results in muramoyl L-Alanine amidase activity but no muramic delta-lactam formation. Expression of PdaA alone in E. coli had no effect on E. coli peptidoglycan structure, whereas expression of CwlD and PdaA together resulted in the formation of muramic delta-lactam. CwlD and PdaA are necessary and sufficient for muramic delta-lactam production, and no other B. subtilis gene product is required. PdaA probably carries out both deacetylation and lactam ring formation and requires the product of CwlD activity as a substrate. 相似文献
3.
Kelly A. Fimlaid Owen Jensen M. Lauren Donnelly Michael B. Francis Joseph A. Sorg Aimee Shen 《PLoS pathogens》2015,11(10)
Clostridium difficile is a Gram-positive spore-forming pathogen and a leading cause of nosocomial diarrhea. C. difficile infections are transmitted when ingested spores germinate in the gastrointestinal tract and transform into vegetative cells. Germination begins when the germinant receptor CspC detects bile salts in the gut. CspC is a subtilisin-like serine pseudoprotease that activates the related CspB serine protease through an unknown mechanism. Activated CspB cleaves the pro-SleC zymogen, which allows the activated SleC cortex hydrolase to degrade the protective cortex layer. While these regulators are essential for C. difficile spores to outgrow and form toxin-secreting vegetative cells, the mechanisms controlling their function have only been partially characterized. In this study, we identify the lipoprotein GerS as a novel regulator of C. difficile spore germination using targeted mutagenesis. A gerS mutant has a severe germination defect and fails to degrade cortex even though it processes SleC at wildtype levels. Using complementation analyses, we demonstrate that GerS secretion, but not lipidation, is necessary for GerS to activate SleC. Importantly, loss of GerS attenuates the virulence of C. difficile in a hamster model of infection. Since GerS appears to be conserved exclusively in related Peptostreptococcaeace family members, our results contribute to a growing body of work indicating that C. difficile has evolved distinct mechanisms for controlling the exit from dormancy relative to B. subtilis and other spore-forming organisms. 相似文献
4.
Ehud Ohana Eitan Hoch Chen Keasar Taiho Kambe Ofer Yifrach Michal Hershfinkel Israel Sekler 《The Journal of biological chemistry》2009,284(26):17677-17686
Vesicular zinc transporters (ZnTs) play a critical role in regulating Zn2+ homeostasis in various cellular compartments and are linked to major diseases ranging from Alzheimer disease to diabetes. Despite their importance, the intracellular localization of ZnTs poses a major challenge for establishing the mechanisms by which they function and the identity of their ion binding sites. Here, we combine fluorescence-based functional analysis and structural modeling aimed at elucidating these functional aspects. Expression of ZnT5 was followed by both accelerated removal of Zn2+ from the cytoplasm and its increased vesicular sequestration. Further, activity of this zinc transport was coupled to alkalinization of the trans-Golgi network. Finally, structural modeling of ZnT5, based on the x-ray structure of the bacterial metal transporter YiiP, identified four residues that can potentially form the zinc binding site on ZnT5. Consistent with this model, replacement of these residues, Asp599 and His451, with alanine was sufficient to block Zn2+ transport. These findings indicate, for the first time, that Zn2+ transport mediated by a mammalian ZnT is catalyzed by H+/Zn2+ exchange and identify the zinc binding site of ZnT proteins essential for zinc transport. 相似文献
5.
Zinc-induced paracrystalline aggregation of glutamine synthetase 总被引:22,自引:0,他引:22
The unique capacity of glutamine synthetase to form highly insoluble paracrystalline aggregates in the presence of Zn2+ and Mg2+ mixtures is the basis of a new simple procedure for the isolation of the enzyme from crude extracts of Escherichia coli. Under optimal conditions (pH 5.85, 25 °C, 1.5 mm ZnSO4 and 50 MgCl2 over 95% of the enzyme is precipitated from crude extracts; differential extraction of the precipitate with dilute buffer (pH 7.0) containing 2.5 mm MgCl2 leads to high yields of almost pure glutamine synthetase. Polyacrylamide gel electrophoresis of the purified enzyme shows it to consist of one major protein and two minor protein components, all of which exhibit glutamine synthetase activity. The major component appears to be identical with the enzyme previously isolated by the older more tedious procedure of Woolfolk et al. (1966). The γ-glutamyl transferase activity of enzyme isolated by the new procedure is the same as that isolated by the older method, but its biosynthetic activity is 25–35% lower. In all other respects examined (i.e., divalent ion specificity, pH optimum, apparent Km values for substrates, susceptibility to feedback inhibition and physical properties) enzymes prepared by the old and the new procedures are indistinguishable. From studies with pure glutamine synthetase isolated by either procedure, it has been established that paracrystalline aggregation does not occur until 9–10 equivs of Zn2+ are bound per mole of enzyme. The high specificity of Zn2+ in inducing enzyme aggregation, suggests that its binding provokes a unique conformational state of the enzyme. This is supported by the fact that addition of Zn2+ to relaxed (divalent cation free) enzyme elicits a change in the ultraviolet spectrum of the enzyme that is qualitatively different from that caused by either Mg2+ or Mn2+. Moreover, in contrast to Mg2+, the binding of Zn2+ decreases the fluorescence associated with the binding of 2-p-toludinyl-naphthalene-6-sulfonic acid to the enzyme, suggesting that Zn2+ binding is accompanied by a decrease in the number of exposed hydrophobic regions on the enzyme. 相似文献
6.
Ekaterina L. Nemashkalova Eugene A. Permyakov Sergei E. Permyakov Ekaterina A. Litus 《Biometals》2017,30(3):341-353
Human serum albumin (HSA) is an abundant multiligand carrier protein, linked to progression of Alzheimer’s disease (AD). Blood HSA serves as a depot of amyloid β (Aβ) peptide. Aβ peptide-buffering properties of HSA depend on interaction with its ligands. Some of the ligands, namely, linoleic acid (LA), zinc and copper ions are involved into AD progression. To clarify the interplay between LA and metal ion binding to HSA, the dependence of LA binding to HSA on Zn2+, Cu2+, Mg2+ and Ca2+ levels and structural consequences of these interactions have been explored. Seven LA molecules are bound per HSA molecule in the absence of the metal ions. Zn2+ binding to HSA causes a loss of one bound LA molecule, while the other metals studied exert an opposite effect (1–2 extra LA molecules are bound). In most cases, the observed effects are not related to the metal-induced changes in HSA quaternary structure. However, the Zn2+-induced decline in LA capacity of HSA could be due to accumulation of multimeric HSA forms. Opposite to Ca2+/Mg2+-binding, Zn2+ or Cu2+ association with HSA induces marked changes in its hydrophobic surface. Overall, the divalent metal ions modulate LA capacity and affinity of HSA to a different extent. LA- and Ca2+-binding to HSA synergistically support each other. Zn2+ and Cu2+ induce more pronounced changes in hydrophobic surface and quaternary structure of HSA and its LA capacity. A misbalanced metabolism of these ions in AD could modify interactions of HSA with LA, other fatty acids and hydrophobic substances, associated with AD. 相似文献
7.
The ability of the bacteriophage-encoded peptidoglycan hydrolases (endolysins) to destroy Gram-positive bacteria from without
makes these enzymes promising antimicrobials. Recombinant endolysins from Listeria monocytogenes phages have been shown to rapidly lyse and kill the pathogen in all environments. To determine optimum conditions regarding
application of recombinant Listeria phage endolysins in food or production equipments, properties of different Listeria endolysins were studied. Optimum NaCl concentration for the amidase HPL511 was 200 nM and 300 mM for the peptidases HPL118,
HPL500, and HPLP35. Unlike most other peptidoglycan hydrolases, all four enzymes exhibited highest activity at elevated pH
values at around pH 8–9. Lytic activity was abolished by EDTA and could be restored by supplementation with various divalent
metal cations, indicating their role in catalytic function. While substitution of the native Zn2+ by Ca2+ or Mn2+ was most effective in case of HPL118, HPL500, and HPLP35, supplementation with Co2+ and Mn2+ resulted in an approximately 5-fold increase in HPL511 activity. Interestingly, the glutamate peptidases feature a conserved
SxHxxGxAxD zinc-binding motif, which is not present in the amidases, although they also require centrally located divalent
metals for activity. The endolysins HPL118, HPL511, and HPLP35 revealed a surprisingly high thermostability, with up to 35%
activity remaining after 30 min incubation at 90°C. The available data suggest that denaturation at elevated temperatures
is reversible and may be followed by rapid refolding into a functional state. 相似文献
8.
Peptidoglycan recognition proteins (PGRPs) are a family of innate immune molecules that recognize bacterial peptidoglycan. PGRPs are highly conserved in invertebrates and vertebrates including fish. However, the biological function of teleost PGRP remains largely uninvestigated. In this study, we identified a PGRP homologue, SoPGLYRP-2, from red drum (Sciaenops ocellatus) and analyzed its activity and potential function. The deduced amino acid sequence of SoPGLYRP-2 is composed of 482 residues and shares 46-94% overall identities with known fish PGRPs. SoPGLYRP-2 contains at the C-terminus a single zinc amidase domain with conserved residues that form the catalytic site. Quantitative RT-PCR analysis detected SoPGLYRP-2 expression in multiple tissues, with the highest expression occurring in liver and the lowest expression occurring in brain. Experimental bacterial infection upregulated SoPGLYRP-2 expression in kidney, spleen, and liver in time-dependent manners. To examine the biological activity of SoPGLYRP-2, purified recombinant proteins representing the intact SoPGLYRP-2 (rSoPGLYRP-2) and the amidase domain (rSoPGLYRP-AD) were prepared from Escherichia coli. Subsequent analysis showed that rSoPGLYRP-2 and rSoPGLYRP-AD (i) exhibited comparable Zn2+-dependent peptidoglycan-lytic activity and were able to recognize and bind to live bacterial cells, (ii) possessed bactericidal effect against Gram-positive bacteria and slight bacteriostatic effect against Gram-negative bacteria, (iii) were able to block bacterial infection into host cells. These results indicate that SoPGLYRP-2 is a zinc-dependent amidase and a bactericide that targets preferentially at Gram-positive bacteria, and that SoPGLYRP-2 is likely to play a role in host innate immune defense during bacterial infection. 相似文献
9.
Ling Fu Xuegong Li Xiang Xiao Jun Xu 《Extremophiles : life under extreme conditions》2014,18(2):429-440
Amidases catalyze the hydrolysis of amides to free carboxylic acids and ammonia. Hyperthermophilic archaea are a natural reservoir of various types of thermostable enzymes. Here, we report the purification and characterization of an amidase from Pyrococcus yayanosii CH1, the first representative of a strict-piezophilic hyperthermophilic archaeon that originated from a deep-sea hydrothermal vent. An open reading frame that encoded a putative member of the nitrilase protein superfamily was identified. We cloned and overexpressed amiE in Escherichia coli C41 (DE3). The purified AmiE enzyme displayed maximal activity at 85 °C and pH 6.0 (NaH2PO4–Na2HPO4) with acetamide as the substrate and showed activity over the pH range of 4–8 and the temperature range of 4–95 °C. AmiE is a dimer and active on many aliphatic amide substrates, such as formamide, acetamide, hexanamide, acrylamide, and l-glutamine. Enzyme activity was induced by 1 mM Ca2+, 1 mM Al3+, and 1–10 mM Mg2+, but strongly inhibited by Zn2+, Cu2+, Ni2+, and Fe3+. The presence of acetone and ethanol significantly decreased the enzymatic activity. Neither 5 % methanol nor 5 % isopropanol had any significant effect on AmiE activity (99 and 96 % retained, respectively). AmiE displayed amidase activity although it showed high sequence homology (78 % identity) with the known nitrilase from Pyrococcus abyssi. AmiE is the most characterized archaeal thermostable amidase in the nitrilase superfamily. The thermostability and pH-stability of AmiE will attract further studies on its potential industrial applications. 相似文献
10.
Magdalena Plotka Anna-Karina Kaczorowska Agnieszka Morzywolek Joanna Makowska Lukasz P. Kozlowski Audur Thorisdottir Sigurlaug Skírnisdottir Sigridur Hj?rleifsdottir Olafur H. Fridjonsson Gudmundur O. Hreggvidsson Jakob K. Kristjansson Slawomir Dabrowski Janusz M. Bujnicki Tadeusz Kaczorowski 《PloS one》2015,10(9)
Phage vB_Tsc2631 infects the extremophilic bacterium Thermus scotoductus MAT2631 and uses the Ts2631 endolysin for the release of its progeny. The Ts2631 endolysin is the first endolysin from thermophilic bacteriophage with an experimentally validated catalytic site. In silico analysis and computational modelling of the Ts2631 endolysin structure revealed a conserved Zn2+ binding site (His30, Tyr58, His131 and Cys139) similar to Zn2+ binding site of eukaryotic peptidoglycan recognition proteins (PGRPs). We have shown that the Ts2631 endolysin lytic activity is dependent on divalent metal ions (Zn2+ and Ca2+). The Ts2631 endolysin substitution variants H30N, Y58F, H131N and C139S dramatically lost their antimicrobial activity, providing evidence for the role of the aforementioned residues in the lytic activity of the enzyme. The enzyme has proven to be not only thermoresistant, retaining 64.8% of its initial activity after 2 h at 95°C, but also highly thermodynamically stable (Tm = 99.82°C, ΔHcal = 4.58 × 104 cal mol-1). Substitutions of histidine residues (H30N and H131N) and a cysteine residue (C139S) resulted in variants aggregating at temperatures ≥75°C, indicating a significant role of these residues in enzyme thermostability. The substrate spectrum of the Ts2631 endolysin included extremophiles of the genus Thermus but also Gram-negative mesophiles, such as Escherichia coli, Salmonella panama, Pseudomonas fluorescens and Serratia marcescens. The broad substrate spectrum and high thermostability of this endolysin makes it a good candidate for use as an antimicrobial agent to combat Gram-negative pathogens. 相似文献
11.
The addition of Tb3+ to apoalkaline phosphatase at pH 8.0 results in the formation of a metalloprotein with an enhanced Tb3+ fluorescence at 492, 545, and 580 nm. The Tb3+ excitation spectrum is most consistent with a process in which energy is transferred from one or more tyrosyl chromophores to the bound lanthanide. An analysis of the fluorescence data under equilibrium conditions yields one Tb3+ binding site per enzyme dimer with a Kn = 0.16 ± 0.02 μm. The Tb3+-alkaline phosphatase complex is not catalytically active nor does it incorporate covalently bound phosphate, but the specific activity of Zn2+-alkaline phosphatase is significantly enhanced in the presence of Tb3+ indicating that this lanthanide mimics Mg2+ in stabilizing the structure of alkaline phosphatase. The fluorescence of the Tb3+-enzyme is found to be quite sensitive to conformational changes which occur upon addition of Zn2+ or substrates. 相似文献
12.
13.
Krunal K. Mehta Elena E. Paskaleva Saba Azizi-Ghannad Daniel J. Ley Martin A. Page Jonathan S. Dordick Ravi S. Kane 《Applied and environmental microbiology》2013,79(19):5899-5906
There continues to be a need for developing efficient and environmentally friendly treatments for Bacillus anthracis, the causative agent of anthrax. One emerging approach for inactivation of vegetative B. anthracis is the use of bacteriophage endolysins or lytic enzymes encoded by bacterial genomes (autolysins) with highly evolved specificity toward bacterium-specific peptidoglycan cell walls. In this work, we performed in silico analysis of the genome of Bacillus anthracis strain Ames, using a consensus binding domain amino acid sequence as a probe, and identified a novel lytic enzyme that we termed AmiBA2446. This enzyme exists as a homodimer, as determined by size exclusion studies. It possesses N-acetylmuramoyl-l-alanine amidase activity, as determined from liquid chromatography-mass spectrometry (LC-MS) analysis of muropeptides released due to the enzymatic digestion of peptidoglycan. Phylogenetic analysis suggested that AmiBA2446 was an autolysin of bacterial origin. We characterized the effects of enzyme concentration and phase of bacterial growth on bactericidal activity and observed close to a 5-log reduction in the viability of cells of Bacillus cereus 4342, a surrogate for B. anthracis. We further tested the bactericidal activity of AmiBA2446 against various Bacillus species and demonstrated significant activity against B. anthracis and B. cereus strains. We also demonstrated activity against B. anthracis spores after pretreatment with germinants. AmiBA2446 enzyme was also stable in solution, retaining its activity after 4 months of storage at room temperature. 相似文献
14.
The glyoxalase pathway is ubiquitously found in all the organisms ranging from prokaryotes to eukaryotes. It acts as a major pathway for detoxification of methylglyoxal (MG), which deleteriously affects the biological system in stress conditions. The first important enzyme of this system is Glyoxalase I (GLYI). It is a metalloenzyme which requires divalent metal ions for its activity. This divalent metal ion can be either Zn2+ as found in most of eukaryotes or Ni2+ as seen in prokaryotes. In the present study, we have found three active GLYI enzymes (AtGLYI2, AtGLYI3 and AtGLYI6) belonging to different metal activation classes coexisting in Arabidopsis thaliana. These enzymes have been found to efficiently complement the GLYI yeast mutants. These three enzymes have been characterized in terms of their activity, metal dependency, kinetic parameters and their role in conferring tolerance to multiple abiotic stresses in E. coli and yeast. AtGLYI2 was found to be Zn2+ dependent whereas AtGLYI3 and AtGLYI6 were Ni2+ dependent. Enzyme activity of Zn2+ dependent enzyme, AtGLYI2, was observed to be exceptionally high (~250–670 fold) as compared to Ni2+ dependent enzymes, AtGLYI3 and AtGLYI6. The activity of these GLYI enzymes correlated well to their role in stress tolerance. Heterologous expression of these enzymes in E. coli led to better tolerance against various stress conditions. This is the first report of a higher eukaryotic species having multiple active GLYI enzymes belonging to different metal activation classes. 相似文献
15.
Giulia Veronesi Simon J. Whitehead Francesco Francia Federico Boscherini Giovanni Venturoli Nick P.J. Cotton 《BBA》2010,1797(4):494-500
Transhydrogenase couples hydride transfer between NADH and NADP+ to proton translocation across a membrane. The binding of Zn2+ to the enzyme was shown previously to inhibit steps associated with proton transfer. Using Zn K-edge X-ray absorption fine structure (XAFS), we report here on the local structure of Zn2+ bound to Escherichia coli transhydrogenase. Experiments were performed on wild-type enzyme and a mutant in which βHis91 was replaced by Lys (βH91K). This well-conserved His residue, located in the membrane-spanning domain of the protein, has been suggested to function in proton transfer, and to act as a ligand of the inhibitory Zn2+. The XAFS analysis has identified a Zn2+-binding cluster formed by one Cys, two His, and one Asp/Glu residue, arranged in a tetrahedral geometry. The structure of the site is consistent with the notion that Zn2+ inhibits proton translocation by competing with H+ binding to the His residues. The same cluster of residues with very similar bond lengths best fits the spectra of wild-type transhydrogenase and βH91K. Evidently, βHis91 is not directly involved in Zn2+ binding. The locus of βHis91 and that of the Zn-binding site, although both on (or close to) the proton-transfer pathway of transhydrogenase, are spatially separate. 相似文献
16.
Zinc (Zn2+) was shown to invariably inhibit muscimol-stimulated36Cl− uptake by synaptoneurosomes in the cerebral cortex, hippocampus and cerebellum. The Zn2+ sensitivity of the GABAA receptor-gated36Cl− uptake in the cerebral cortex was comparable to that in the hippocampus, whereas the uptake in the cerebellum was less sensitive
to Zn2+. Although diazepam-potentiation of muscimol-stimulated36Cl− uptake was unaltered by 100 μM Zn2+ in the cerebellum. Zn2+ inhibited [3H]diazepam binding significantly at 1 mM in the cerebral cortex and cerebellum, whereas Ni2+ increased the binding in a concentration-dependent manner in both regions. Although lower concentrations of Zn2+ did not affect [3H]Ro 15-4513 binding to diazepam-sensitive sites, higher concentrations of Zn2+ increased the binding in both regions. Unlike the diazepam-sensitive sites the diazepam-insensitive [3H]Ro 15-4513 binding was not affected by Zn2+ or Ni2+ at any of the tested concentrations. These results suggest that the GABAA ligand-gated Cl− flux and its diazepam-potentiation are heterogeneously modulated in various brain regions. It is also suggested that cerebellar
diazepam-insensitive [3H]Ro 15-4513 binding sites are insensitive to Zn2+ and Ni2+. 相似文献
17.
Prosenjit Sen Sanghamitra Sahoo Usha R. Pendurthi L. Vijaya Mohan Rao 《The Journal of biological chemistry》2010,285(26):20410-20420
Zinc is an essential trace element for human nutrition and is critical to the structure, stability, and function of many proteins. Zinc ions were shown to enhance activation of the intrinsic pathway of coagulation but down-regulate the extrinsic pathway of coagulation. The protein C pathway plays a key role in blood coagulation and inflammation. At present there is no information on whether zinc modulates the protein C pathway. In the present study we found that Zn2+ enhanced the binding of protein C/activated protein C (APC) to endothelial cell protein C receptor (EPCR) on endothelial cells. Binding kinetics revealed that Zn2+ increased the binding affinities of protein C/APC to EPCR. Equilibrium dialysis with 65Zn2+ revealed that Zn2+ bound to the Gla domain as well as sites outside of the Gla domain of protein C/APC. Intrinsic fluorescence measurements suggested that Zn2+ binding induces conformational changes in protein C/APC. Zn2+ binding to APC inhibited the amidolytic activity of APC, but the inhibition was reversed by Ca2+. Zn2+ increased the rate of APC generation on endothelial cells in the presence of physiological concentrations of Ca2+ but did not further enhance increased APC generation obtained in the presence of physiological concentrations of Mg2+ with Ca2+. Zn2+ had no effect on the anticoagulant activity of APC. Zn2+ enhanced APC-mediated activation of protease activated receptor 1 and p44/42 MAPK. Overall, our data show that Zn2+ binds to protein C/APC, which results in conformational changes in protein C/APC that favor their binding to EPCR. 相似文献
18.
Effects of multivalent cations on cell wall-associated Acid phosphatase activity 总被引:2,自引:2,他引:0
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Primary cell walls, free from cytoplasmic contamination were prepared from corn (Zea mays L.) roots and potato (Solanum tuberosum) tubers. After EDTA treatment, the bound acid phosphatase activities were measured in the presence of various multivalent cations. Under the conditions of minimized Donnan effect and at pH 4.2, the bound enzyme activity of potato tuber cell walls (PCW) was stimulated by Cu2+, Mg2+, Zn2+, and Mn2+; unaffected by Ba2+, Cd2+, and Pb2+; and inhibited by Al3+. The bound acid phosphatase of PCW was stimulated by a low concentration but inhibited by a higher concentration of Hg2+. On the other hand, in the case of corn root cell walls (CCW), only inhibition of the bound acid phosphatase by Al3+ and Hg2+ was observed. Kinetic analyses revealed that PCW acid phosphatase exhibited a negative cooperativity under all employed experimental conditions except in the presence of Mg2+. In contrast, CCW acid phosphatase showed no cooperative behavior. The presence of Ca2+ significantly reduced the effects of Hg2+ or Al3+, but not Mg2+, to the bound cell wall acid phosphatases. The salt solubilized (free) acid phosphatases from both PCW and CCW were not affected by the presence of tested cations except for Hg2+ or Al3+ which caused a Ca2+-insensitive inhibition of the enzymes. The induced stimulation or inhibition of bound acid phosphatases was quantitatively related to cation binding in the cell wall structure. 相似文献
19.
Hloise Coullon Aline Rifflet Richard Wheeler Claire Janoir Ivo G. Boneca Thomas Candela 《The Journal of biological chemistry》2020,295(49):16785
Clostridium difficile is an anaerobic and spore-forming bacterium responsible for 15–25% of postantibiotic diarrhea and 95% of pseudomembranous colitis. Peptidoglycan is a crucial element of the bacterial cell wall that is exposed to the host, making it an important target for the innate immune system. The C. difficile peptidoglycan is largely N-deacetylated on its glucosamine (93% of muropeptides) through the activity of enzymes known as N-deacetylases, and this N-deacetylation modulates host–pathogen interactions, such as resistance to the bacteriolytic activity of lysozyme, virulence, and host innate immune responses. C. difficile genome analysis showed that 12 genes potentially encode N-deacetylases; however, which of these N-deacetylases are involved in peptidoglycan N-deacetylation remains unknown. Here, we report the enzymes responsible for peptidoglycan N-deacetylation and their respective regulation. Through peptidoglycan analysis of several mutants, we found that the N-deacetylases PdaV and PgdA act in synergy. Together they are responsible for the high level of peptidoglycan N-deacetylation in C. difficile and the consequent resistance to lysozyme. We also characterized a third enzyme, PgdB, as a glucosamine N-deacetylase. However, its impact on N-deacetylation and lysozyme resistance is limited, and its physiological role remains to be dissected. Finally, given the influence of peptidoglycan N-deacetylation on host defense against pathogens, we investigated the virulence and colonization ability of the mutants. Unlike what has been shown in other pathogenic bacteria, a lack of N-deacetylation in C. difficile is not linked to a decrease in virulence. 相似文献
20.
Lei Sun Peter Vella Robert Schnell Anna Polyakova Gleb Bourenkov Fengyang Li Annika Cimdins Thomas R. Schneider Ylva Lindqvist Michael Y. Galperin Gunter Schneider Ute Römling 《Journal of molecular biology》2018,430(18):3170-3189
Many bacteria secrete cellulose, which forms the structural basis for bacterial multicellular aggregates, termed biofilms. The cellulose synthase complex of Salmonella typhimurium consists of the catalytic subunits BcsA and BcsB and several auxiliary subunits that are encoded by two divergently transcribed operons, bcsRQABZC and bcsEFG. Expression of the bcsEFG operon is required for full-scale cellulose production, but the functions of its products are not fully understood. This work aimed to characterize the BcsG subunit of the cellulose synthase, which consists of an N-terminal transmembrane fragment and a C-terminal domain in the periplasm. Deletion of the bcsG gene substantially decreased the total amount of BcsA and cellulose production. BcsA levels were partially restored by the expression of the transmembrane segment, whereas restoration of cellulose production required the presence of the C-terminal periplasmic domain and its characteristic metal-binding residues. The high-resolution crystal structure of the periplasmic domain characterized BcsG as a member of the alkaline phosphatase/sulfatase superfamily of metalloenzymes, containing a conserved Zn2+-binding site. Sequence and structural comparisons showed that BcsG belongs to a specific family within alkaline phosphatase-like enzymes, which includes bacterial Zn2+-dependent lipopolysaccharide phosphoethanolamine transferases such as MCR-1 (colistin resistance protein), EptA, and EptC and the Mn2+-dependent lipoteichoic acid synthase (phosphoglycerol transferase) LtaS. These enzymes use the phospholipids phosphatidylethanolamine and phosphatidylglycerol, respectively, as substrates. These data are consistent with the recently discovered phosphoethanolamine modification of cellulose by BcsG and show that its membrane-bound and periplasmic parts play distinct roles in the assembly of the functional cellulose synthase and cellulose production. 相似文献