首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 46 毫秒
1.
LigD 3′-phosphoesterase (PE) enzymes perform end-healing reactions at DNA breaks. Here we characterize the 3′-ribonucleoside-resecting activity of Candidatus Korarchaeum PE. CkoPE prefers a single-stranded substrate versus a primer–template. Activity is abolished by vanadate (10 mM), but is less sensitive to phosphate (IC50 50 mM) or chloride (IC50 150 mM). The metal requirement is satisfied by manganese, cobalt, copper or cadmium, but not magnesium, calcium, nickel or zinc. Insights to CkoPE metal specificity were gained by solving new 1.5 Å crystal structures of CkoPE in complexes with Co2+ and Zn2+. His9, His15 and Asp17 coordinate cobalt in an octahedral complex that includes a phosphate anion, which is in turn coordinated by Arg19 and His51. The cobalt and phosphate positions and the atomic contacts in the active site are virtually identical to those in the CkoPE·Mn2+ structure. By contrast, Zn2+ binds in the active site in a tetrahedral complex, wherein the position, orientation and atomic contacts of the phosphate are shifted and its interaction with His51 is lost. We conclude that: (i) PE selectively binds to ‘soft’ metals in either productive or non-productive modes and (ii) PE catalysis depends acutely on proper metal and scissile phosphate geometry.  相似文献   

2.
Aminoacylase 3 (AA3) deacetylates N-acetyl-aromatic amino acids and mercapturic acids including N-acetyl-1,2-dichlorovinyl-L-cysteine (Ac-DCVC), a metabolite of a xenobiotic trichloroethylene. Previous studies did not demonstrate metal-dependence of AA3 despite a high homology with a Zn2+-metalloenzyme aminoacylase 2 (AA2). A 3D model of mouse AA3 was created based on homology with AA2. The model showed a putative metal binding site formed by His21, Glu24 and His116, and Arg63, Asp68, Asn70, Arg71, Glu177 and Tyr287 potentially involved in catalysis/substrate binding. The mutation of each of these residues to alanine inactivated AA3 except Asn70 and Arg71, therefore the corrected 3D model of mouse AA3 was created. Wild type (wt) mouse AA3 expressed in E. coli contained ∼ 0.35 zinc atoms per monomer. Incubation with Co2+ and Ni2+ activated wt-AA3. In the cobalt-activated AA3 zinc was replaced with cobalt. Metal removal completely inactivated wt-AA3, whereas addition of Zn2+, Mn2+ or Fe2+ restored initial activity. Co2+ and to a lesser extent Ni2+ increased activity several times in comparison with intact wt-AA3. Co2+ drastically increased the rate of deacetylation of Ac-DCVC and significantly increased the toxicity of Ac-DCVC in the HEK293T cells expressing wt-AA3. The results indicate that AA3 is a metalloenzyme significantly activated by Co2+ and Ni2+.  相似文献   

3.
Energy-coupling factor (ECF) transporters for vitamins and metal ions in prokaryotes consist of two ATP-binding cassette-type ATPases, a substrate-specific transmembrane protein (S component) and a transmembrane protein (T component) that physically interacts with the ATPases and the S component. The mechanism of ECF transporters was analyzed upon reconstitution of a bacterial biotin transporter into phospholipid bilayer nanodiscs. ATPase activity was not stimulated by biotin and was only moderately reduced by vanadate. A non-hydrolyzable ATP analog was a competitive inhibitor. As evidenced by cross-linking of monocysteine variants and by site-specific spin labeling of the Q-helix followed by EPR-based interspin distance analyses, closure and reopening of the ATPase dimer (BioM2) was a consequence of ATP binding and hydrolysis, respectively. A previously suggested role of a stretch of small hydrophobic amino acid residues within the first transmembrane segment of the S units for S unit/T unit interactions was structurally and functionally confirmed for the biotin transporter. Cross-linking of this segment in BioY (S) using homobifunctional thiol-reactive reagents to a coupling helix of BioN (T) indicated a reorientation rather than a disruption of the BioY/BioN interface during catalysis. Fluorescence emission of BioY labeled with an environmentally sensitive fluorophore was compatible with an ATP-induced reorientation and consistent with a hypothesized toppling mechanism. As demonstrated by [3H]biotin capture assays, ATP binding stimulated substrate capture by the transporter, and subsequent ATP hydrolysis led to substrate release. Our study represents the first experimental insight into the individual steps during the catalytic cycle of an ECF transporter in a lipid environment.  相似文献   

4.
Energy-coupling factor (ECF) transporters form a distinct group of ABC-type micronutrient importers in prokaryotes that do not contain extracytoplasmic, soluble substrate-binding proteins. Instead, they consist of a transmembrane substrate-specific S component that interacts with a module composed of a moderately conserved transmembrane (T) component and ABC ATPases. The majority of S components is considered to act as high-affinity binding proteins that strictly depend on their cognate T and ATPase units for transport activity. For a fraction of biotin-specific S units, however, transport activity was demonstrated in their solitary state. Here, we compared the activities of nickel- and cobalt-specific ECF transporters in the presence and absence of their T and ATPase units. Accumulation assays with radioactive metal ions showed that the truncated transporters led to approx. 25 % of cell-bound radioactivity compared to the holotransporters. Activity of urease, an intracellular nickel-dependent enzyme, was used as a reporter and clearly indicated that the cell-bound radioactivity correlates with the cytoplasmic metal concentration. The results demonstrate that S units of metal transporters not only bind their substrates on the cell surface but mediate transport across the membrane, a finding of general importance on the way to understand the mechanism of ECF transporters.  相似文献   

5.
ATP-binding cassette (ABC) transporters of the cluster 9 family are ubiquitous among bacteria and essential for acquiring Zn2+ and Mn2+ from the environment or, in the case of pathogens, from the host. These rely on a substrate-binding protein (SBP) to coordinate the relevant metal with high affinity and specificity and subsequently release it to a membrane permease for translocation into the cytoplasm. Although a number of cluster 9 SBP structures have been determined, the structural attributes conferring Zn2+ or Mn2+ specificity remain ambiguous. Here we describe the gene expression profile, in vitro metal binding properties, and crystal structure of a new cluster 9 SBP from Paracoccus denitrificans we have called AztC. Although all of our results strongly indicate Zn2+ over Mn2+ specificity, the Zn2+ ion is coordinated by a conserved Asp residue only observed to date as a metal ligand in Mn2+-specific SBPs. The unusual sequence properties of this protein are shared among close homologues, including members from the human pathogens Klebsiella pneumonia and Enterobacter aerogenes, and would seem to suggest a subclass of Zn2+-specific transporters among the cluster 9 family. In any case, the unusual coordination environment of AztC expands the already considerable range of those available to Zn2+-specific SBPs and highlights the presence of a His-rich loop as the most reliable indicator of Zn2+ specificity.  相似文献   

6.
Energy-coupling factor (ECF) transporters, a recently discovered class of importers of micronutrients, are composed of a substrate-specific transmembrane component (S component) and a conserved energy-coupling module consisting of a transmembrane protein (T component) and pairs of ABC ATPases (A proteins). Based on utilization of a dedicated (subclass I) or shared (subclass II) energy-coupling module, ECF systems fall into two subclasses. The T components are the least-characterized proteins of ECF importers, and their function is essentially unknown. Using RcBioN and LmEcfT, the T units of the subclass I biotin transporter (RcBioMNY) of a gram-negative bacterium and of the subclass II folate, pantothenate, and riboflavin transporters of a lactic acid bacterium, respectively, we analyzed the role of two strongly conserved short motifs, each containing an arginine residue. Individual replacement of the two Arg residues in RcBioN reduced ATPase activity, an indicator of the transporter function, by two-thirds without affecting the modular assembly of the RcBioMNY complex. A double Arg-to-Glu replacement destroyed the complex and abolished ATPase activity. The corresponding single mutation in motif II of LmEcfT, as well as a double mutation, led to loss of the T unit from the subclass II ECF transporters and inactivated these systems. A single Arg-to-Glu replacement in motif I, however, abolished vitamin uptake activity without affecting assembly of the modules. Our results indicate that the conserved motif I in T components is essential for intramolecular signaling and, in cooperation with motif II, for subunit assembly of modular ECF transporters.Energy-coupling factor (ECF) transporters are a recently discovered novel class of importers of micronutrients in prokaryotes (9, 12, 13, 20). They are composed of a conserved energy-coupling module consisting of a transmembrane protein (T component) and pairs of ATP-binding cassette-containing proteins (A proteins), as well as an S unit (S component) through which substrate specificity is conveyed. S components represent a group of highly diverse small integral membrane proteins with predicted or experimentally established individual specificity for transition metal ions, B vitamins or their precursors, biotin, lipoate, and intermediates of salvage pathways. A link between substrate specificity and S components has been experimentally demonstrated in the case of CbiMN (Co2+) and NikMN (Ni2+) (13), RibU (riboflavin) (4, 6, 19), BioY (biotin) (9), FolT (folate) (8, 12), and ThiT (thiamine) (8, 12, 14). Based on utilization of a dedicated or shared AAT module, ECF transporters fall into two subclasses. Members of subclass I are encoded by operons containing one or two ABC ATPase genes, a T-component gene, and an S-component gene (12). RcBioMNY, the biotin transporter of Rhodobacter capsulatus, is the prototype of these systems. Previous work has established that the solitary BioY (S component) can function as a low-affinity transporter. It is converted into a high-affinity system in the presence of the AAT module BioMN (9). Notably, the majority of ECF transporters belong to subclass II. These promiscuous systems are widespread in the Firmicutes and also occur in members of the Thermotogales lineage and in archaea (12). In general, the cells contain a single ecfA1A2T operon and a number of genes for S units that are scattered around the genome. Cooccurrence of subclass I and subclass II ECF transporters is found in archaeal and bacterial species. The bioinformatic prediction for subclass II systems suggesting that several diverse S components interact with the same EcfA1A2T module has recently been confirmed experimentally for folate, riboflavin, and thiamine transporters of Bacillus subtilis, Lactobacillus casei, and Leuconostoc mesenteroides and for the hypothetical pantothenate transporter of L. mesenteroides (12).The modular composition of ECF transporters poses questions about their oligomeric structure, the specificity of subunit recognition, and the intersubunit signaling that couples substrate uptake to ATP hydrolysis by the ABC ATPase domains. These issues are essentially unsolved for any ECF transporter.In the present study, we confirm the predicted role of L. mesenteroides PanT (LmPanT) as a pantothenate-specific S component that functionally interacts with the LmEcfA1A2T module. The main focus is on the role of the T components, which are the least-characterized proteins of ECF importers. T proteins have moderately similar primary structures. Two 3-amino-acid signatures with Ala-Arg-Gly as the consensus sequence in the C-terminal part are the most conserved feature in the T units. Using RcBioMNY as a member of subclass I and LmEcfA1A2T plus LmFolT, LmPanT, or LmRibU as representatives of subclass II ECF transporters, we obtained evidence that replacement of either of the Arg residues in the T proteins strongly reduces or abolishes activity of the systems. While double mutations interfered with complex stability in each case, single replacements of the Arg residue in motif I did not have a marked impact on stability of the complexes, suggesting that this residue is important primarily for intramolecular signaling in both subclasses of ECF transporters.  相似文献   

7.
nhlF and hoxN, the genes encoding a cobalt transporter of Rhodococcus rhodochrous J1 and a nickel permease of Alcaligenes eutrophus H16, respectively, were expressed in Escherichia coli. 57Co2+ and 63Ni2+ transport of the recombinants was examined by means of a previously described physiological assay. Although the transporters are highly similar, different preferences for divalent transition metal cations were observed. HoxN was unable to transport 57Co2+, but mediated 63Ni2+ uptake. The latter activity was unaffected by a tenfold excess of other divalent cations, showing the specificity of HoxN for Ni2+. In contrast, NhlF transported both 57Co2+ and 63Ni2+ ion. NhlF-mediated 63Ni2+ uptake was markedly reduced in the presence of Co2+, while 57Co2+ uptake was only slightly lower in the presence of Ni2+. These results indicate different affinities of NhlF for Co2+ and Ni2+ and identified Co2+ ion as the preferred substrate. Received: 8 September 1998 / Accepted: 30 November 1998  相似文献   

8.
ZnuA is the periplasmic Zn2+-binding protein associated with the high-affinity ATP-binding cassette ZnuABC transporter from Escherichia coli. Although several structures of ZnuA and its homologs have been determined, details regarding metal ion stoichiometry, affinity, and specificity as well as the mechanism of metal uptake and transfer remain unclear. The crystal structures of E. coli ZnuA (Eco-ZnuA) in the apo, Zn2+-bound, and Co2+-bound forms have been determined. ZnZnuA binds at least two metal ions. The first, observed previously in other structures, is coordinated tetrahedrally by Glu59, His60, His143, and His207. Replacement of Zn2+ with Co2+ results in almost identical coordination geometry at this site. The second metal binding site involves His224 and several yet to be identified residues from the His-rich loop that is unique to Zn2+ periplasmic metal binding receptors. Electron paramagnetic resonance and X-ray absorption spectroscopic data on CoZnuA provide additional insight into possible residues involved in this second site. The second site is also detected by metal analysis and circular dichroism (CD) titrations. Eco-ZnuA binds Zn2+ (estimated K d < 20 nM), Co2+, Ni2+, Cu2+, Cu+, and Cd2+, but not Mn2+. Finally, conformational changes upon metal binding observed in the crystal structures together with fluorescence and CD data indicate that only Zn2+ substantially stabilizes ZnuA and might facilitate recognition of ZnuB and subsequent metal transfer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.  相似文献   

9.
The peptidases in clan MH are known as cocatalytic zinc peptidases that have two zinc ions in the active site, but their metal preference has not been rigorously investigated. In this study, the molecular basis for metal preference is provided from the structural and biochemical analyses. Kinetic studies of Pseudomonas aeruginosa aspartyl aminopeptidase (PaAP) which belongs to peptidase family M18 in clan MH revealed that its peptidase activity is dependent on Co2+ rather than Zn2+: the kcat (s−1) values of PaAP were 0.006, 5.10 and 0.43 in no-metal, Co2+, and Zn2+ conditions, respectively. Consistently, addition of low concentrations of Co2+ to PaAP previously saturated with Zn2+ greatly enhanced the enzymatic activity, suggesting that Co2+ may be the physiologically relevant cocatalytic metal ion of PaAP. The crystal structures of PaAP complexes with Co2+ or Zn2+ commonly showed two metal ions in the active site coordinated with three conserved residues and a bicarbonate ion in a tetragonal geometry. However, Co2+- and Zn2+-bound structures showed no noticeable alterations relevant to differential effects of metal species, except the relative orientation of Glu-265, a general base in the active site. The characterization of mutant PaAP revealed that the first metal binding site is primarily responsible for metal preference. Similar to PaAP, Streptococcus pneumonia glutamyl aminopeptidase (SpGP), belonging to aminopeptidase family M42 in clan MH, also showed requirement for Co2+ for maximum activity. These results proposed that clan MH peptidases might be a cocatalytic cobalt peptidase rather than a zinc-dependent peptidase.  相似文献   

10.
Transhydrogenase couples proton translocation across a bacterial or mitochondrial membrane to the redox reaction between NAD(H) and NADP(H). Purified intact transhydrogenase from Escherichia coli was prepared, and its His tag removed. The forward and reverse transhydrogenation reactions catalysed by the enzyme were inhibited by certain metal ions but a “cyclic reaction” was stimulated. Of metal ions tested they were effective in the order Pb2+ > Cu2+ > Zn2+ = Cd2+ > Ni2+ > Co2+. The results suggest that the metal ions affect transhydrogenase by binding to a site in the proton-transfer pathway. Attenuated total-reflectance Fourier-transform infrared difference spectroscopy indicated the involvement of His and Asp/Glu residues in the Zn2+-binding site(s). A mutant in which βHis91 in the membrane-spanning domain of transhydrogenase was replaced by Lys had enzyme activities resembling those of wild-type enzyme treated with Zn2+. Effects of the metal ion on the mutant were much diminished but still evident. Signals in Zn2+-induced FTIR difference spectra of the βHis91Lys mutant were also attributable to changes in His and Asp/Glu residues but were much smaller than those in wild-type spectra. The results support the view that βHis91 and nearby Asp or Glu residues participate in the proton-transfer pathway of transhydrogenase.  相似文献   

11.
In this study, bioaccumulation and heavy metal resistance of Cd2+, Cu2+, Co2+ and Mn2+ ions by thermophilic Geobacillus thermantarcticus and Anoxybacillus amylolyticus was investigated. The bacteria, in an order with respect to metal resistance from the most resistant to the most sensitive, was found to be Mn2+ > Co2+ > Cu2+ > Cd2+ for both G. thermantarcticus and A. amylolyticus. It was determined that the highest metal bioaccumulation was performed by A. amylolyticus in Mn2+ (28,566 μg/g dry weight), and the lowest metal bioaccumulation was performed by A. amylolyticus in Co2+ (327.3 μg/g dry weight). The highest Cd2+ capacities of dried cell membrane was found to be 36.07 and 39.55 mg/g membrane for G. thermantarticus and A. amylolyticus, respectively, and the highest Cd2+ capacities of wet cell membrane was found to be 14.36 and 12.39 mg/g membrane for G. thermantarcticus and A. amylolyticus, respectively.  相似文献   

12.
Pnkp is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from many phyla. Pnkp is composed of three catalytic modules: an N-terminal polynucleotide 5′ kinase, a central 2′,3′ phosphatase and a C-terminal ligase. The phosphatase module is a Mn2+-dependent phosphodiesterase–monoesterase that dephosphorylates 2′,3′-cyclic phosphate RNA ends. Here we report the crystal structure of the phosphatase domain of Clostridium thermocellum Pnkp with Mn2+ and citrate in the active site. The protein consists of a core binuclear metallo-phosphoesterase fold (exemplified by bacteriophage λ phosphatase) embellished by distinctive secondary structure elements. The active site contains a single Mn2+ in an octahedral coordination complex with Asp187, His189, Asp233, two citrate oxygens and a water. The citrate fills the binding site for the scissile phosphate, wherein it is coordinated by Arg237, Asn263 and His264. The citrate invades the site normally occupied by a second metal (engaged by Asp233, Asn263, His323 and His376), and thereby dislocates His376. A continuous tract of positive surface potential flanking the active site suggests an RNA binding site. The structure illuminates a large body of mutational data regarding the metal and substrate specificity of Clostridium thermocellum Pnkp phosphatase.  相似文献   

13.
Complex formation properties of a novel water soluble thiazolyloxime 2-(4-methylthiazol-2-yl)-2-(hydroxyimino)acetic acid (H3L1) with Cu2+ and Ni2+ were investigated in solution by potentiometrical and spectral (UV-Vis, EPR, NMR) methods. All Cu2+ and most of Ni2+ complex species detected in solution were found to have square-planar MN4 core with oxime and heterocyclic nitrogen atoms which was rationalized in terms of destabilizing effect of repulsive interaction between oxygen atom of carboxylic group and nitrogen atom of thiazole ring in N,O-coordinated ligand conformation. It has been found that stability of metal complexes in a series of oxime ligands is dependent upon basicity of nitrogen atom of oxime group. The thiazolyloxime forms less stable complexes with Cu2+ but stronger ones with Ni2+ ions when compared to parent 2-(hydroxyimino)propanoic acid. The lower stability obtained for Cu2+ complexes was elucidated in terms of negative inductive effect of the thiazole and nitrile substituents as well as an effect of intramolecular attractive interaction between thiazolyl sulfur and oxime oxygen atoms in thiazolyloxime. In the case of Ni2+ the complexes formed are square-planar and it is why thiazolyl ligand is more effective in metal ion binding than simple 2-(hydroxyimino)propanoic acid forming only octahedral species. The solid state structure of the Co3+ complex K3[Co(HL1)3]·5.5H2O (1) was studied by X-ray analysis. The thiazolyloxime ligand is coordinated to Co3+ via oxime nitrogen and carboxylate oxygen atoms forming five-membered chelate rings.  相似文献   

14.
The kinetics of the binding of Co2+ to apoalkaline phosphatase from Escherichia coli have been followed by stopped flow spectroscopy of the tyrosines and bound Co2+, revealing three well-separated kinetic phases with time constants in the milliseconds, seconds, and minutes time ranges. Four Co2+ are bound per dimer judging from the saturation behavior of an isomerization reaction linked to a rapid metal binding step. These Co2+ are distributed in pairs to each of two different types of binding sites distinguishable by their participation in two different isomerization-linked transients in the seconds and minutes time ranges, respectively. Two of the observed isomerizations are tentatively assigned to subtle protein conformational changes since they are reflected in both the Co2+ and aromatic amino acid chromophores but not by changes in ORD, CD, viscosity, or ultracentrifugation properties. They may therefore represent local changes in regions of the protein near the metal sites.  相似文献   

15.
Helicobacter pylori UreF (HpUreF) is involved in the insertion of Ni2+ in the urease active site. The recombinant protein in solution is a dimer characterized by an extensive α-helical structure and a well-folded tertiary structure. HpUreF binds two Ni2+ ions per dimer, with a micromolar dissociation constant, as shown by calorimetry. X-ray absorption spectroscopy indicated that the Ni2+ ions reside in a five-coordinate pyramidal geometry comprising exclusively N/O-donor ligands derived from the protein, including one or two histidine imidazole and carboxylate ligands. Binding of Ni2+ does not affect the solution properties of the protein. Mutation to alanine of His229 and/or Cys231, a pair of residues located on the protein surface that interact with H. pylori UreD, altered the affinity of the protein for Ni2+. This result, complemented by the findings from X-ray absorption spectroscopy, indicates that the Ni2+ binding site involves His229, and that Cys231 has an indirect structural role in metal binding. An in vivo assay of urease activation demonstrated that H229A HpUreF, C231A HpUreF, and H229/C231 HpUreF are significantly less competent in this process, suggesting a role for a Ni2+ complex with UreF in urease maturation. This hypothesis was supported by calculations revealing the presence of a tunnel that joins the Cys-Pro-His metal binding site on UreG and an opening on the UreD surface, passing through UreF close to His229 and Cys231, in the structure of the H. pylori UreDFG complex. This tunnel could be used to transfer nickel into the urease active site during apoenzyme-to-holoenzyme activation.  相似文献   

16.
The interaction of cobalt (Co2+) and nickel (Ni2+) ions with whole cells of the photosynthetic purple bacterium Rhodobacter sphaeroides strain R26 was investigated. Active and passive uptakes were examined in cells grown in the presence of increasing amounts of Co2+ and Ni2+. Inductively coupled plasma atomic emission spectroscopy (ICP-AES), pH titration, and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy were used to assess the role of cell envelope and metabolism in accumulating the two heavy metals. The chosen microorganism was able to uptake cobalt and nickel up to 2.2 and 0.25 mg per gram of dried cells respectively, with the largest part found bound to the cell surface. Carboxylate groups lying on the cell wall of this Gram-negative bacterium proved to be the major candidates for binding protons and metal cations. Co2+ was found to interfere with Mg2+ extracellular immobilization and transport across the membrane, indicating that these ions share binding sites on the cell envelope and ion transport systems. According to the presence of a competition mechanism, bacterial growth experiments showed that high Mg2+ concentrations are able to rescue R. sphaeroides from Co2+ toxicity.  相似文献   

17.
Bacterial allantoinase (ALLase; EC 3.5.2.5), which catalyzes the conversion of allantoin into allantoate, possesses a binuclear metal center in which two metal ions are bridged by a posttranslationally carboxylated lysine. Here, we characterized ALLase from Escherichia coli BL21. Purified recombinant ALLase exhibited no activity but could be activated when preincubating with some metal ions before analyzing its activity, and was in the order: Mn2+- ≫ Co2+- > Zn2+- > Ni2+- > Cd2+- ~Mg2+-activated enzyme; however, activity of ALLase (Mn2+-activated form) was also significantly inhibited with 5 mM Co2+, Zn2+, and Cd2+ ions. Activity of Mn2+-activated ALLase was increased by adding the reducing agent dithiothreitol (DTT), but was decreased by treating with the sulfhydryl modifying reagent N-ethylmaleimide (NEM). Inhibition of Mn2+-activated ALLase by chelator 8-hydroxy-5-quinolinesulfonic acid (8-HQSA), but not EDTA, was pH-dependent. Analysis of purified ALLase by gel filtration chromatography revealed a mixture of monomers, dimers, and tetramers. Substituting the putative metal binding residues His59, His61, Lys146, His186, His242, and Asp315 with Ala completely abolished the activity of ALLase, even preincubating with Mn2+ ions. On the basis of these results, as well as the pH-activity profile, the reaction mechanism of ALLase is discussed and compared with those of other cyclic amidohydrolases.  相似文献   

18.
Peptide deformylase (PDF, E.C. 3.5.1.88) catalyzes the removal of N-terminal formyl groups from nascent ribosome-synthesized polypeptides. PDF contains a catalytically essential divalent metal ion, which is tetrahedrally coordinated by three protein ligands (His, His, and Cys) and a water molecule. Previous studies revealed that the metal cofactor is a Fe2+ ion in Escherichia coli and many other bacterial PDFs. In this work, we found that PDFs from two iron-deficient bacteria, Borrelia burgdorferi and Lactobacillus plantarum, are stable and highly active under aerobic conditions. The native B. burgdorferi PDF (BbPDF) was purified 1200-fold and metal analysis revealed that it contains ∼1.1 Zn2+ ion/polypeptide but no iron. Our studies suggest that PDF utilizes different metal ions in different organisms. These data have important implications in designing PDF inhibitors and should help address some of the unresolved issues regarding PDF structure and catalytic function.  相似文献   

19.
Plants have developed numerous strategies to cope with phosphorus (P) deficiency resulting from low availability in soils. Evolution of ethylene and up-regulation of root secreted acid phosphatase activity are common for plants in response to P deficiency. To determine the role of ethylene in response of plants to P deficiency, we investigated the effects of ethylene precursor (1-amino cyclopropane-1-carboxylic acid, ACC) and ethylene synthesis antagonists (aminoethoxyvinylglycine AVG, cobalt, Co2+) on P concentrations in roots and shoots of Medicago falcata seedlings grown in P-sufficient (500 μM H2PO4) and P-deficient (5 μM H2PO4) solution. After transferring M. falcata seedlings from P-sufficient to P-deficient solution for 2 days, root P concentration was significantly reduced. The reduction in root P concentration was reversed by AVG and Co2+, and a similar reduction in root P concentration of seedlings exposed to P-sufficient solution was observed by ACC. Expression of high-affinity phosphate transporters (MfPT1, MfPT5) was enhanced by P-deficiency and this process was reversed by AVG and Co2+. There was a marked increase in activity of root acid phosphatase (APase) and expression of gene encoding APase (MfPAP1) under P-deficient conditions, and the increase in APAse activity and expression of MfPAP1 was inhibited by AVG and Co2+. APase activity and expression of MfPAP1 expression in seedlings grown in P-sufficient solution were enhanced by ACC. Root and shoot P concentrations were increased when organic phosphorus was added to the P-deficient solution, and the increase in P concentration was significantly inhibited by AVG and Co2+. These results indicate that ethylene plays an important role in modulation of P acquisition by possibly mobilizing organic P via up-regulating root APase activity and high-affinity phosphate transporters.  相似文献   

20.
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号