Identification of three shikimate kinase genes in rice: characterization of their differential expression during panicle development and of the enzymatic activities of the encoded proteins

The shikimate pathway is common to the biosynthesis of the three aromatic amino acids and that of various secondary metabolites in land plants. Shikimate kinase (SK; EC 2.7.1.71) catalyzes the phosphorylation of shikimate to yield shikimate 3-phosphate. In an attempt to elucidate the functional roles of enzymes that participate in the shikimate pathway in rice (Oryza sativa), we have now identified and characterized cDNAs corresponding to three SK genes—OsSK1, OsSK2, and OsSK3—in this monocotyledenous plant. These SK cDNAs encode proteins with different NH₂-terminal regions and with putative mature regions that share sequence similarity with other plant and microbial SK proteins. An in vitro assay of protein import into intact chloroplasts isolated from pea (Pisum sativum) seedlings revealed that the full-length forms of the three rice SK proteins are translocated into chloroplasts and processed, consistent with the assumption that the different NH₂-terminal sequences function as chloroplast transit peptides. The processed forms of all three rice proteins synthesized in vitro manifested SK catalytic activity. Northern blot analysis revealed that the expression of OsSK1 and OsSK2 was induced in rice calli by treatment with the elicitor N-acetylchitoheptaose, and that expression of OsSK1 and OsSK3 was up-regulated specifically during the heading stage of panicle development. These results suggest that differential expression of the three rice SK genes and the accompanying changes in the production of shikimate 3-phosphate may contribute to the defense response and to panicle development in rice.The nucleotide sequences of OsSK1, OsSK2, and OsSK3 cDNAs are available in GenBank under the accession numbers AB188834, AB188835, and AB188836, respectively.     

Heterologous expression and characterization of the sterol 14α-demethylase CYP51F1 from Candida albicans

Lanosterol 14α-demethylase (CYP51F1) from Candida albicans is known to be an essential enzyme in fungal sterol biosynthesis. Wild-type CYP51F1 and several of its mutants were heterologously expressed in Escherichia coli, purified, and characterized. It exhibited a typical reduced CO-difference spectrum with a maximum at 446 nm. Reconstitution of CYP51F1 with NADPH-P450 reductase gave a system that successfully converted lanosterol to its demethylated product. Titration of the purified enzyme with lanosterol produced a typical type I spectral change with K_d = 6.7 μM. The azole antifungal agents econazole, fluconazole, ketoconazole, and itraconazole bound tightly to CYP51F1 with K_d values between 0.06 and 0.42 μM. The CYP51F1 mutations F105L, D116E, Y132H, and R467K frequently identified in clinical isolates were examined to determine their effect on azole drug binding affinity. The azole K_d values of the purified F105L, D116E, and R467K mutants were little altered. A homology model of C. albicans CYP51F1 suggested that Tyr132 in the BC loop is located close to the heme in the active site, providing a rationale for the modified heme environment caused by the Y132H substitution. Taken together, functional expression and characterization of CYP51F1 provide a starting basis for the design of agents effective against C. albicans infections.     

Structural basis for shikimate-binding specificity of Helicobacter pylori shikimate kinase

Shikimate kinase (EC 2.7.1.71) catalyzes the specific phosphorylation of the 3-hydroxyl group of shikimic acid in the presence of ATP. As the fifth key step in the shikimate pathway for aromatic amino acid biosynthesis in bacteria, fungi, and plants, but not mammals, shikimate kinase represents an attractive target for the development of new antimicrobial agents, herbicides, and antiparasitic agents. Here, we report the 1.8-Angstroms crystal structure of Helicobacter pylori shikimate kinase (HpSK). The crystal structure shows a three-layer alpha/beta fold consisting of a central sheet of five parallel beta-strands flanked by seven alpha-helices. An HpSK-shikimate-PO(4) complex was also determined and refined to 2.3 Angstroms, revealing induced-fit movement from an open to a closed form on substrate binding. Shikimate is located above a short 3(10) helix formed by a strictly conserved motif (GGGXV) after beta(3). Moreover, several highly conserved charged residues including Asp33 (in a conserved DT/SD motif), Arg57, and Arg132 (interacting with shikimate) are identified, guiding the development of novel inhibitors of shikimate kinase.     

Discovery of Helicobacter pylori shikimate kinase inhibitors: bioassay and molecular modeling

Shikimate kinase (SK) is the fifth enzyme in the shikimate pathway and catalyzes the phosphate transfer from ATP to shikimate in generating shikimate 3-phosphate and ADP. SK has been developed as a promising target for the discovery of antibacterial agents. In this report, two small molecular inhibitors (compound 1, 3-methoxy-4-{[2-({2-methoxy-4-[(4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]phenoxy}methyl)benzyl]oxy}benzaldehyde; compound 2, 5-bromo-2-(5-{[1-(3,4-dichlorophenyl)-3,5-dioxo-4-pyrazolidinylidene]methyl}-2-furyl)benzoic acid) against Helicobacter pylori SK (HpSK) were successfully identified with IC(50) values of 5.5+/-1.2 and 6.4+/-0.4 microM, respectively. The inhibition kinetics shows that compound 1 is a noncompetitive inhibitor with respect to both shikimate and MgATP, and compound 2 is a competitive inhibitor toward shikimate and noncompetitive inhibitor with respect to MgATP. The surface plasmon resonance (SPR) technology based analysis reveals that the equilibrium dissociation constants (K(D)s) of compounds 1 and 2 with HpSK enzyme are 4.39 and 3.74 microM, respectively. The molecular modeling and docking of two inhibitors with HpSK reveals that the active site of HpSK is rather roomy and deep, forming an L-shape channel on the surface of the protein, and compound 1 prefers the corner area of L-shape channel, while compound 2 binds the short arm of the channel of SK in the binding interactions. It is expected that our current work might supply useful information for the development of novel SK inhibitors.     

GHRH antagonist when combined with cytotoxic agents induces S-phase arrest and additive growth inhibition of human colon cancer

Treatment of colon cancer with an antagonist of growth hormone-releasing hormone (GHRH), JMR-132, results in a cell cycle arrest in S-phase of the tumor cells. Thus, we investigated the effect of JMR-132 in combination with S-phase-specific cytotoxic agents, 5-FU, irinotecan and cisplatin on the in vitro and in vivo growth of HT-29, HCT-116 and HCT-15 human colon cancer cell lines. In vitro, every compound inhibited proliferation of HCT-116 cells in a dose-dependent manner. Treatment with JMR-132 (5 μM) combined with 5-FU (1.25 μM), irinotecan (1.25 μM) or cisplatin (1.25 μM) resulted in an additive growth inhibition of HCT-116 cells in vitro as shown by MTS assay. Cell cycle analyses revealed that treatment of HCT-116 cells with JMR-132 was accompanied by a cell cycle arrest in S-phase. Combination treatment using JMR-132 plus a cytotoxic drug led to a significant increase of the sub-G₁ fraction, suggesting apoptosis. In vivo, daily treatment with GHRH antagonist JMR-132 decreased the tumor volume by 40–55% (p < 0.001) of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice. Combined treatment with JMR-132 plus chemotherapeutic agents 5-FU, irinotecan or cisplatin resulted in an additive tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts to 56–85%. Our observations indicate that JMR-132 enhances the antiproliferative effect of S-phase-specific cytotoxic drugs by causing accumulation of tumor cells in S-phase.     

Crystal structure of Mycobacterium tuberculosis shikimate kinase in complex with shikimic acid and an ATP analogue

Shikimate kinase (SK) and other enzymes in the shikimate pathway are potential targets for developing nontoxic antimicrobial agents, herbicides, and antiparasite drugs, because the pathway is essential in microorganisms, plants, and parasites but absent from mammals. SK catalyzes the reaction of phosphoryl transfer from ATP to shikimic acid (SA). Since 2002, a total of 11 SK structures have been reported, but none contains either the two substrate (SA and ATP) or the two product (SA-phosphate and ADP) molecules. Here, we present three crystal structures of SK from Mycobacterium tuberculosis (MtSK), including apo-MtSK, a binary complex MtSK x SA, and the ternary complex of MtSK with SA and an ATP analogue, AMPPCP. The structures of apo-MtSK and MtSK x AMPPCP x SA make it possible to elucidate the conformational changes of MtSK upon the binding of both substrates; the structure of MtSK x AMPPCP x SA reveals interactions between the protein and gamma-phosphate which indicate dynamic roles of catalytic residues Lys15 and Arg117.     

GHRH antagonist when combined with cytotoxic agents induces S-phase arrest and additive growth inhibition of human colon cancer

Treatment of colon cancer with an antagonist of growth hormone-releasing hormone (GHRH), JMR-132, results in a cell cycle arrest in S-phase of the tumor cells. Thus, we investigated the effect of JMR-132 in combination with S-phase-specific cytotoxic agents, 5-FU, irinotecan and cisplatin on the in vitro and in vivo growth of HT-29, HCT-116 and HCT-15 human colon cancer cell lines. In vitro, every compound inhibited proliferation of HCT-116 cells in a dose-dependent manner. Treatment with JMR-132 (5 μM) combined with 5-FU (1.25 μM), irinotecan (1.25 μM) or cisplatin (1.25 μM) resulted in an additive growth inhibition of HCT-116 cells in vitro as shown by MTS assay. Cell cycle analyses revealed that treatment of HCT-116 cells with JMR-132 was accompanied by a cell cycle arrest in S-phase. Combination treatment using JMR-132 plus a cytotoxic drug led to a significant increase of the sub-G₁ fraction, suggesting apoptosis. In vivo, daily treatment with GHRH antagonist JMR-132 decreased the tumor volume by 40–55% (p < 0.001) of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice. Combined treatment with JMR-132 plus chemotherapeutic agents 5-FU, irinotecan or cisplatin resulted in an additive tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts to 56–85%. Our observations indicate that JMR-132 enhances the antiproliferative effect of S-phase-specific cytotoxic drugs by causing accumulation of tumor cells in S-phase.     

The RNR motif of B. subtilis RNase P protein interacts with both PRNA and pre-tRNA to stabilize an active conformer

Ribonuclease P (RNase P) catalyzes the metal-dependent 5′ end maturation of precursor tRNAs (pre-tRNAs). In Bacteria, RNase P is composed of a catalytic RNA (PRNA) and a protein subunit (P protein) necessary for function in vivo. The P protein enhances pre-tRNA affinity, selectivity, and cleavage efficiency, as well as modulates the cation requirement for RNase P function. Bacterial P proteins share little sequence conservation although the protein structures are homologous. Here we combine site-directed mutagenesis, affinity measurements, and single turnover kinetics to demonstrate that two residues (R60 and R62) in the most highly conserved region of the P protein, the RNR motif (R60–R68 in Bacillus subtilis), stabilize PRNA complexes with both P protein (PRNA•P protein) and pre-tRNA (PRNA•P protein•pre-tRNA). Additionally, these data indicate that the RNR motif enhances a metal-stabilized conformational change in RNase P that accompanies substrate binding and is essential for efficient catalysis. Stabilization of this conformational change contributes to both the decreased metal requirement and the enhanced substrate recognition of the RNase P holoenzyme, illuminating the role of the most highly conserved region of P protein in the RNase P reaction pathway.     

Free tyrosine and tyrosine-rich peptide-dependent superoxide generation catalyzed by a copper-binding,threonine-rich neurotoxic peptide derived from prion protein

Previously, generation of superoxide anion (O₂^•-) catalyzed by Cu-binding peptides derived from human prion protein (model sequence for helical Cu-binding motif VNITKQHTVTTTT was most active) in the presence of catecholamines and related aromatic monoamines such as phenylethylamine and tyramine, has been reported [Kawano, T., Int J Biol Sci 2007; 3: 57-63]. The peptide sequence (corresponding to helix 2) tested here is known as threonine-rich neurotoxic peptide. In the present article, the redox behaviors of aromatic monoamines, 20 amino acids and prion-derived tyrosine-rich peptide sequences were compared as putative targets of the oxidative reactions mediated with the threonine-rich prion-peptide. For detection of O₂^•-, an O₂^•--specific chemiluminescence probe, Cypridina luciferin analog was used. We found that an aromatic amino acid, tyrosine (structurally similar to tyramine) behaves as one of the best substrates for the O₂^•- generating reaction (conversion from hydrogen peroxide) catalyzed by Cu-bound prion helical peptide. Data suggested that phenolic moiety is required to be an active substrate while the presence of neither carboxyl group nor amino group was necessarily required. In addition to the action of free tyrosine, effect of two tyrosine-rich peptide sequences YYR and DYEDRYYRENMHR found in human prion corresponding to the tyrosine-rich region was tested as putative substrates for the threonine-rich neurotoxic peptide. YYR motif (found twice in the Y-rich region) showed 2- to 3-fold higher activity compared to free tyrosine. Comparison of Y-rich sequence consisted of 13 amino acids and its Y-to-F substitution mutant sequence revealed that the tyrosine-residues on Y-rich peptide derived from prion may contribute to the higher production of O₂^•-. These data suggest that the tyrosine residues on prion molecules could be additional targets of the prion-mediated reactions through intra- or inter-molecular interactions. Lastly, possible mechanism of O₂^•- generation and the impacts of such self-redox events on the conformational changes in prion are discussed.     

Crystal structure of shikimate kinase from Mycobacterium tuberculosis reveals the dynamic role of the LID domain in catalysis

Shikimate kinase (SK) and other enzymes in the shikimate pathway are potential targets for developing non-toxic antimicrobial agents, herbicides, and anti-parasite drugs, because the pathway is essential in the above species but is absent from mammals. The crystal structure of Mycobacterium tuberculosis SK (MtSK) in complex with MgADP has been determined at 1.8 A resolution, revealing critical information for the structure-based design of novel anti-M. tuberculosis agents. MtSK, with a five-stranded parallel beta-sheet flanked by eight alpha-helices, has three domains: the CORE domain, the shikimate-binding domain (SB), and the LID domain. The ADP molecule is bound with its adenine moiety sandwiched between the side-chains of Arg110 and Pro155, its beta-phosphate group in the P-loop, and the alpha and beta-phosphate groups hydrogen bonded to the guanidinium group of Arg117. Arg117 is located in the LID domain, is strictly conserved in SK sequences, is observed for the first time to interact with any bound nucleotide, and appears to be important in both substrate binding and catalysis. The crystal structure of MtSK (this work) and that of Erwinia chrysanthemi SK suggest a concerted conformational change of the LID and SB domains upon nucleotide binding.     

Evolutionary Diversification of Plant Shikimate Kinase Gene Duplicates

Shikimate kinase (SK; EC 2.7.1.71) catalyzes the fifth reaction of the shikimate pathway, which directs carbon from the central metabolism pool to a broad range of secondary metabolites involved in plant development, growth, and stress responses. In this study, we demonstrate the role of plant SK gene duplicate evolution in the diversification of metabolic regulation and the acquisition of novel and physiologically essential function. Phylogenetic analysis of plant SK homologs resolves an orthologous cluster of plant SKs and two functionally distinct orthologous clusters. These previously undescribed genes, shikimate kinase-like 1 (SKL1) and -2 (SKL2), do not encode SK activity, are present in all major plant lineages, and apparently evolved under positive selection following SK gene duplication over 400 MYA. This is supported by functional assays using recombinant SK, SKL1, and SKL2 from Arabidopsis thaliana (At) and evolutionary analyses of the diversification of SK-catalytic and -substrate binding sites based on theoretical structure models. AtSKL1 mutants yield albino and novel variegated phenotypes, which indicate SKL1 is required for chloroplast biogenesis. Extant SKL2 sequences show a strong genetic signature of positive selection, which is enriched in a protein–protein interaction module not found in other SK homologs. We also report the first kinetic characterization of plant SKs and show that gene expression diversification among the AtSK inparalogs is correlated with developmental processes and stress responses. This study examines the functional diversification of ancient and recent plant SK gene duplicates and highlights the utility of SKs as scaffolds for functional innovation.     

Metabolic engineering of Corynebacterium glutamicum for shikimate overproduction by growth-arrested cell reaction

Corynebacterium glutamicum with the ability to simultaneously utilize glucose/pentose mixed sugars was metabolically engineered to overproduce shikimate, a valuable hydroaromatic compound used as a starting material for the synthesis of the anti-influenza drug oseltamivir. To achieve this, the shikimate kinase and other potential metabolic activities for the consumption of shikimate and its precursor dehydroshikimate were inactivated. Carbon flux toward shikimate synthesis was enhanced by overexpression of genes for the shikimate pathway and the non-oxidative pentose phosphate pathway. Subsequently, to improve the availability of the key aromatics precursor phosphoenolpyruvate (PEP) toward shikimate synthesis, the PEP: sugar phosphotransferase system (PTS) was inactivated and an endogenous myo-inositol transporter IolT1 and glucokinases were overexpressed. Unexpectedly, the resultant non-PTS strain accumulated 1,3-dihydroxyacetone (DHA) and glycerol as major byproducts. This observation and metabolome analysis identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-catalyzed reaction as a limiting step in glycolysis. Consistently, overexpression of GAPDH significantly stimulated both glucose consumption and shikimate production. Blockage of the DHA synthesis further improved shikimate yield. We applied an aerobic, growth-arrested and high-density cell reaction to the shikimate production by the resulting strain and notably achieved the highest shikimate titer (141 g/l) and a yield (51% (mol/mol)) from glucose reported to date after 48 h in minimal medium lacking nutrients required for cell growth. Moreover, comparable shikimate productivity could be attained through simultaneous utilization of glucose, xylose, and arabinose, enabling efficient shikimate production from lignocellulosic feedstocks. These findings demonstrate that C. glutamicum has significant potential for the production of shikimate and derived aromatic compounds.     

Contribution of each Trp residue toward the intrinsic fluorescence of the Giα1 protein

G_iα1 is the inhibitory G-protein that, upon activation, reduces the activity of adenylyl cyclase. Comparison of the crystal structures of G_iα1 bound to GDP•AMF or GTPγS with that of the inactive, GPD-bound protein indicates that a conformational change occurs in the activation step centered on three switch regions. The contribution of each tryptophan residue (W211 in the switch II region, W131 in the α-helical domain, and W258 in the GTPase domain) toward the intrinsic protein fluorescence was evaluated by using W211F, W131F, and W258F mutants. All three tryptophan residues contributed significantly toward the emission spectra regardless of the conformation. When activated by either GDP•AMF or GTPγS, the observed maximal-fluorescence scaled according to the solvent accessibilities of the tryptophan residues, calculated from molecular dynamics simulations. In the GDP•AMF and GTPγS, but not in the GDP, conformations, the residues W211 and R208 are in close proximity and form a π-cation interaction that results in a red shift in the emission spectra of WT, and W131F and W258F mutants, but a blue shift for the W211F mutant. The observed shifts did not show a relationship with the span of the W211-R208 bridge, but rather with changes in the total interaction energies. Trypsin digestion of the active conformations only occurred for the W211F mutant indicating that the electrostatic π-cation interaction blocks access to R208, which was consistent with the molecular dynamics simulations. We conclude that solvent accessibility and interaction energies account for the fluorescence features of G_iα1.     

The shikimate pathway regulates programmed cell death

Programmed cell death (PCD) is essential for both plant development and stress responses including immunity. However, how plants control PCD is not well-understood. The shikimate pathway is one of the most important metabolic pathways in plants, but its relationship to PCD is unknown. Here, we show that the shikimate pathway promotes PCD in Arabidopsis. We identify a photoperiod-dependent lesion-mimic mutant named Lesion in short-day (lis), which forms spontaneous lesions in short-day conditions. Map-based cloning and whole-genome resequencing reveal that LIS encodes MEE32, a bifunctional enzyme in the shikimate pathway. Metabolic analysis shows that the level of shikimate is dramatically increased in lis. Through genetic screenings, three suppressors of lis (slis) are identified and the causal genes are cloned. SLISes encode proteins upstream of MEE32 in the shikimate pathway. Furthermore, exogenous shikimate treatment causes PCD. Our study uncovers a link between the shikimate pathway and PCD, and suggests that the accumulation of shikimate is an alternative explanation for the action of glyphosate, the most successful herbicide.     

Diversity and Antioxidant Activity of Culturable Endophytic Fungi from Alpine Plants of Rhodiola crenulata,R. angusta,and R. sachalinensis

Rhodiola spp. are rare and endangered alpine plants widely used as medicines and food additives by many civilizations since ancient times. Their main effective ingredients (such as salidroside and p-tyrosol) are praised to exhibit pharmacologic effects on high-altitude sickness and possess anti-aging and other adaptogenic capacities based on their antioxidant properties. In this study, 347 endophytic fungi were isolated from R. crenulata, R. angusta, and R. sachalinensis, and the molecular diversity and antioxidant activities of these fungi were investigated for the first time. These fungi were categorized into 180 morphotypes based on cultural characteristics, and their rRNA gene ITS sequences were analyzed by BLAST search in the GenBank database. Except for 12 unidentified fungi (6.67%), all others were affiliated to at least 57 genera in 20 orders of four phyla, namely, Ascomycota (88.89%), Basidiomycota (2.78%), Zygomycota (1.11%), and Glomeromycota (0.56%), which exhibited high abundance and diversity. Antioxidant assay showed that the DPPH radical-scavenging rates of 114 isolates (63.33%) were >50%, and those of five isolates (Rct45, Rct63, Rct64, Rac76, and Rsc57) were >90%. The EC50 values of five antioxidant assays suggested significant potential of these fungi on scavenging DPPH•, O₂−•, and OH• radicals, as well as scavenging nitrite and chelating Fe²⁺, which showed preference and selection between endophytic fungi and their hosts. Further research also provided the first evidence that Rac12 could produce salidrosides and p-tyrosol. Results suggested that versatile endophytic fungi associated with Rhodiola known as antioxidants could be exploited as potential sources of novel antioxidant products.     

Synthesis and separation of tritium-labeled intermediates of the shikimate pathway

[5-³H]Shikimate (sp radioact 2000 Ci/mol) has been synthesized by reduction of the methyl ester of 5-dehydroshikimate with NaB³H₄ and subsequent hydrolysis of the ester group (M. M. Leduc, P. M. Dansette, and R. G. Azerad (1970) Eur. J. Biochem.15, 428–435). The [5-³H]shikimate has been converted enzymatically to [5-³H]chorismate and [5-³H]prephenate of similar high specific radioactivity by using a cell-free extract of Aerobacter aerogenes 62-1. In addition, a chromatograhic procedure, which utilizes polyethyleneimine-cellulose thin-layer chromatograms, has been developed for the separation of intermediates along the shikimate pathway between shikimate and hydroxyphenylpyruvate or phenylpyruvate. Since the method allows quantitative measurement of tritium-labeled intermediates, it provides the basis for sensitive radioassays of the individual enzymes and allows study of the reaction flux along the overall pathway. The same intermediates can be separated on a large scale by use of a column of DEAE-Sephacel.     

Effect of 6-thioguanine on the stability of duplex DNA

The incorporation of 6-thioguanine (S6G) into DNA is a prerequisite for its cytotoxic action, but duplex structure is not significantly perturbed by the presence of the lesion [J. Bohon and C. R. de los Santos (2003) Nucleic Acids Res., 31, 1331–1338]. It is therefore possible that the mechanism of cytotoxicity relies on a loss of stability rather than a pathway involving direct structural recognition. The research described here focuses on the changes in thermodynamic properties of duplex DNA owing to the introduction of S6G as well as the kinetic properties of base pairs involving S6G. Replacement of a guanine in a G•C pair by S6G results in ~1 kcal/mol less favorable Gibbs free energy of duplex formation at 37°C. S6G•T and G•T mismatch-containing duplexes have almost identical Gibbs free energy at 37°C, with values ~3 kcal/mol less favorable than that of the control. Base pair stability is affected by S6G. The lifetime of the normal G•C base pair is ~125 ms, whereas that of the G•T mismatch is below the detection limit. The lifetimes of S6G•C and S6G•T pairs are ~7 and 2 ms, respectively, demonstrating that, although S6G significantly decreases the stability of the pairing with cytosine, it slightly increases that of a mismatch.     

The potential physiological implications of polygalacturonic acid-mediated production of superoxide

PGA/OGA/PF represent apoplastic signaling molecules implicated in the control of gene expression and the activity of enzymes involved in defense regulation. However, the underlying mechanisms behind such processes are lacking. Here we unequivocally show using EPR spectroscopy with DEPMPO spin-trap capable of differentiating between ^•OH and ^•O₂⁻ that PGA and PF can produce ^•O₂⁻ by transforming ^•OH. The potential physiological implications of this unique property are discussed. We propose that PGA/OGA/PF could represent the initiators of redox signaling cascades in stress response, with H₂O₂ being a downstream secondary messenger.Key words: polygalacturonic acid, pectin, superoxide, hydrogen peroxide, apoplast, redox signalingPGA/OGA/PF represent apoplastic signaling molecules involved in defense regulation.^1–5 For example, they induce de novo enzyme synthesis in the wound-inducible defense reaction⁶ and increase the resistance of plants to pathogens.⁷ However, the underlying mechanisms behind such processes are lacking. Aldington et al.⁸ have postulated that OGAs do not act through a receptor, but rather they owe their activity to some specific physical property. Pertinent to this is the fact that there is a broad range of active OGA structures.⁵ In addition, it has been reported that methylated OGAs are not able to trigger signaling pathways that are activated by OGAs possessing ‘free’ carboxyl groups.^9,10 In contrast to this concept, several research groups have showed that PGA/OGA/PF bind to wall-associated kinases (WAK1 and WAK2).^11–15 However, potential effects of PGA/OGA/PF on the activity of WAK1 or WAK2 have not been observed to date. We propose here that the specific property proposed by Aldington and co-workers,⁸ is in fact the ability of the polymers of galacturonic acid (PGA/OGA/PF) to produce ^•O₂⁻. By taking into account previously proposed mechanisms of reaction of PGA with ^•OH,^16,17 and thermodynamic properties of species potentially involved in the reaction,¹⁸ we hypothesized that PGA could transform the ^•OH radical into ^•O₂⁻. To test our hypothesis we investigated the effects of PGA and pectin on radical production in two different ^•OH-generating systems using EPR spectroscopy with the DEPMPO spin-trap capable of differentiating between ^•OH and ^•O₂⁻.¹⁹The results presented in Figure 1 document the ability of PGA to transform ^•OH to ^•O₂⁻. In addition, our experimental approach showed that pectin shares the ^•O₂⁻-producing ability of its constituent PGA. In the control Fenton system (Fe²⁺ + H₂O₂ → ^•OH + OH⁻ + Fe³⁺) only ^•OH radical was produced (Fig. 1A). However in the presence of PGA or pectin, a significant production of ^•O₂⁻ was detected. Haber-Weiss-like reaction (^•O₂⁻ + H₂O₂ → ^•OH + OH⁻ + O₂) generated ^•OH radical, accompanied by a low level of ^•O₂⁻ (Fig. 1B). The supplementation of PGA or pectin to this system led to sole or pronounced production of ^•O₂⁻, respectively. Under the same experimental settings, no ^•O₂⁻ production was observed for other two major extracellular carbohydrates—cellulose and mannan (Fig. 2).Open in a separate windowFigure 1The ability of PGA and pectin to transform ^•OH radical into ^•O₂⁻. Presented are characteristic EPR spectra of adducts of DEPMPO with the ^•OH radical (/OH) and the ^•O₂⁻ radical (/ooh) in two ^•OH-generating systems: (A) Fenton reaction; (B) Haber-Weiss-like reaction; in the absence (control) or presence of PGA or pectin (15 mgml⁻¹ final concentration). The downward triangle represents the characteristic line of the/OH adduct. The circular symbol represents the characteristic line of the/OOH adduct. The grey lines represent the spectral simulations based on signals of DEPMPO adducts contributing to each spectrum in specific percentages [mean values from four experiments (standard deviations were <5%)].Open in a separate windowFigure 2Characteristic EPR spectra of adducts of DEPMPO with the ^•OH radical (downward triangle) and the ^•O₂⁻ radical (circular symbol) in Haber-Weiss-like ^•OH generating system in the absence (control) or presence of cellulose or mannan (15 mgml⁻¹ final concentration). No ^•O₂⁻ production can be observed in the presence of cellulose or mannan.Presented results illustrate the ability of PGA and pectin to transform ^•OH radical into ^•O₂⁻. Other carbohydrates involved in plant metabolism, such as cellulose and mannan, but also glucose and fructose,²⁰ do not show such properties. In addition, methylated PGA do not produce ^•O₂⁻ in the reaction with ^•OH, but methane,²¹ probably with ^•CH₃ radical as an intermediate.²² This implies that carboxyl groups which are characteristic for PGA play a critical role in the production of ^•O₂⁻. Zegota¹⁶ has proposed that pectin and ^•OH react to produce pectin C(5) radical, which further reacts with molecular oxygen thus forming C(5) peroxyl radical. This radical is unstable, especially at physiological pH values,¹⁷ hence it is further decomposed to carbohydrate fragment(s) and superoxide, via ^•O₂⁻-elimination.^16,17Under in vivo settings, superoxide generated in the apoplast by PGA/OGA/PF can be further dismutated by SOD to H₂O₂, which represents a crucial signaling molecule in plants.^23,24 It is very interesting that signaling properties of H₂O₂ in the plant immune response remarkably overlap with the events initiated by OGA: (1) Similarly to the inverted H₂O₂ gradient across the plant plasma membrane,²⁴ OGA has been reported to activate calcium-dependent protein kinases,²⁵ to provoke membrane depolarization with H⁺ influx and K⁺ efflux,²⁶ and to induce activation of mitogen-activated protein kinases.²⁷ (2) Both, OGA^10,28 and H₂O₂^29–31 provoke an influx of Ca²⁺ from the apoplast into the intracellular compartment. (3) It has been documented that apoplastic generation of ^•O₂⁻ and H₂O₂ follows mechanical stress and the recognition of pathogens,^4,32–34 but also the supplementation of OGAs.^35–37 In addition, it has been reported that the supplementation of OGA to plant cells leads to the increase of apoplastic and total concentration of H₂O₂.^37,38 The enlisted results obtained by others and data presented here imply that PGA/OGA/PF could represent the initiators of redox signaling cascades in stress response, with H₂O₂ being a downstream secondary messenger.Hereby-proposed mechanism of apoplastic production of H₂O₂ by PGA/OGA/PF and SOD, depends on ^•OH radicals. Hence, the central question of our hypothesis is: “Where do apoplastic ^•OH radicals come from, under in vivo conditions?”. Hydrogen peroxide is continually generated in the apoplast by NAD(P) H oxidase/SOD, cell wall peroxidase and other sources during normal metabolism, as reviewed by Neill and co-workers.²⁴ The physiological concentrations of H₂O₂ in plants are not well established,⁴⁰ but it seems that apoplastic and total (fresh weight) concentrations are similar and maintained at around 1 µM.^38–40 In the extracellular compartment, continuous generation of H₂O₂ is balanced by its degradation in ^•OH-generating Fenton reaction which involves redox active metals, such as copper and iron.⁴¹ In principle, ^•OH radicals are further removed by apoplastic ascorbate or cell wall constituents (Fig. 3A).^41–43 However, mechanical wounding (e.g., provoked by cold⁴⁴), degradation of the cell wall by pathogenic enzymes (such as polygalacturonase or pectate lyase⁴⁵) or insect chewing could release PGA/OGA/PF from the cell wall into the apoplast. The presence of PGA/OGA/PF in the apoplast related with these events could drastically change apoplastic redox poise. Positively charged redox active metals readily bind to negatively charged polymers of galacturonic acid.⁴⁶ The close proximity of PGA/OGA/PF to the site of ^•OH production could change the fate of ^•OH. Instead of being scavenged, ^•OH could react with PGA/OGA/PF, which leads to ^•O₂⁻ production and subsequent H₂O₂ re-generation (Fig. 3B). Such ‘recycling’ of H₂O₂ could result in a higher steady-state H₂O₂ concentration in the apoplast and consequent H₂O₂ influx, as H₂O₂ is capable of passing the membrane via passive diffusion and specific aquaporins.⁴⁷ In the stress response, H₂O₂ can traverse the membrane, induce Ca²⁺ influx or diffuse into surrounding healthy tissue to modulate enzyme activity⁴⁸ and initiate gene expression,^23,24,49 crucial for subsequent phases of defense and adaptation. To conclude, PGA/OGA/PF could provide the cell with information about the status of the cell wall affected by stressors, via H₂O₂ signaling.Open in a separate windowFigure 3Schematic presentation of potential effects of PGA/OGA/PF released from stressed wall on apoplastic redox poise and H₂O₂ and Ca²⁺ signaling cascades. (A) Redox processes in apoplast under physiological settings. (B) Redox processes in the apoplast of plant cell exposed to stress. PGA/OGA/PF are released from the cell wall into the apoplast changing the redox poise by transforming ^•OH to ^•O₂⁻ and H₂O₂ (‘H₂O₂ recycling’). This could lead to H₂O₂ accumulation, H₂O₂ influx (via diffusion or peroxiporins) or the activation of Ca²⁺ influx, which leads to the activation of different intracellular responses.     

Sth132I,a novel class-IIS restriction endonuclease of Streptococcus thermophilus ST132

The Sth132I restriction endonuclease (R.Sth132I) was detected in Streptococcus thermophilus ST132 and purified to near homogeneity by heparin Sepharose CL-6B affinity chromatography. Fragments from Sth132I digestion of plasmid DNA were subcloned into pUC19 in Escherichia coli DH5α and sequenced. Sequence analysis of inserts and their ligation junction sites revealed that Sth132I is a novel class-IIS restriction endonuclease, which recognizes the non-palindromic sequence5′-CCCG(N)₄-3′3′-GGGC(N)₈-5′.