Influence of metal ions on bioremediation activity of protocatechuate 3,4-dioxygenase from Stenotrophomonas maltophilia KB2

The aim of this paper was to describe the effect of various metal ions on the activity of protocatechuate 3,4-dioxygenase from Stenotrophomonas maltophilia KB2. We also compared activity of different dioxygenases isolated from this strain, in the presence of metal ions, after induction by various aromatic compounds. S. maltophilia KB2 degraded 13 mM 3,4-dihydroxybenzoate, 10 mM benzoic acid and 12 mM phenol within 24 h of incubation. In the presence of dihydroxybenzoate and benzoate, the activity of protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase was observed. Although Fe³⁺, Cu²⁺, Zn²⁺, Co²⁺, Al³⁺, Cd²⁺, Ni²⁺ and Mn²⁺ ions caused 20–80 % inhibition of protocatechuate 3,4-dioxygenase activity, the above-mentioned metal ions (with the exception of Ni²⁺) inhibited catechol 1,2-dioxygenase to a lesser extent or even activate the enzyme. Retaining activity of at least one of three dioxygenases from strain KB2 in the presence of metal ions makes it an ideal bacterium for bioremediation of contaminated areas.     

Purification and characterization of a laccase from Coprinopsis cinerea in Pichia pastoris

A modified laccase gene, CcLCC6, from Coprinopsis cinerea was chemically synthesized according to the yeast codon bias and expressed in Pichia pastoris. The main properties of laccase, effects of ions and inhibitors, and optimal condition for decolouring malachite green (MG) were investigated in this study. The optimal pH level and temperature of laccase are 3.0 and 40 °C, respectively. The metal ions Mn²⁺, Zn²⁺, Fe³⁺ and Al³⁺ could inhibit laccase activity, as well as 1 mM of sodium dodecyl sulphate and sodium thiosulphate. 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), as a mediator, was necessary in decolorizing MG. The optimal pH and temperature for MG decolorization were 3.0 and 50 °C, respectively. Approximately 0.02 μM recombinant laccase could effectively decolour 0.05 mM of MG in 1 h. CcLCC6I could inhibit the toxicity of MG to P. pastoris. This is the first report on the successful expression in P. pastoris of CcLCC6I and its enzymatic property. Laccase can also be considered as a candidate for treating industrial effluent containing MG.     

Hymenolepis diminuta: Partial characterization of the membrane-bound and solubilized alkaline phosphohydrolase activities of the isolated brush border plasma membrane

The membrane-bound and solubilized (using Triton ×-100 or sodium dodecyl sulfate (SDS)) alkaline phosphohydrolase (APase) activities of the isolated brush border membrane of Hymenolepis diminuta require a divalent cation for maximum activity. Highest rates of substrate (p-nitrophenyl phosphate) hydrolysis are obtained with low concentrations of Mg²⁺ (1 mM), although low concentrations of Mn²⁺, Ca²⁺, or Zn²⁺ will also partially satisfy this requirement; higher concentrations of Mg²⁺ and Mn²⁺, and other divalent cations (Cu²⁺, Fe²⁺, and Pb²⁺), inhibit the membrane-bound APase activity. Solubilization of the membrane-bound enzyme in either Triton or SDS results in an increase in specific activity and K_m, but has little effect on thermal stability of the APase activity. Phosphate, pyrophosphate, adenosine 5′-triphosphate, adenosine 5′-monophosphate, glucose 1-phosphate, glucose 6-phosphate, fructose 6-phosphate, and fructose 1,6-diphosphate inhibit substrate hydrolysis, and the relative affinities of these inhibitors for the APase enzyme are altered only slightly upon solubilization. Graphic analyses of data from inhibitor studies indicate that all eight inhibitors will inhibit membrane-bound and solubilized APase activities 100% at high inhibitonsubstrate ratios. Molybdate, F^?, 2-mercaptoethanol, cysteine, and p-chloromercuribenzoate inhibit membrane-bound APase activity. Inhibitor data indicate that if more than one enzyme is responsible for the APase activity of the brush border membrane of H. diminuta, the enzymes cannot be differentiated on the basis of substrate specificity.     

Factors modulating alkaline phosphatase activity in the diazotrophic rice-field cyanobacterium, Anabaena oryzae

Summary Alkaline phosphatases (APase), both phosphomonoesterase (PMEase) and phosphodiesterase (PDEase) were studied in the cyanobacterium Anabaena oryzae for their specific requirements of temperature, pH, micro- and macronutrients and their activities in the presence of salinity and heavy metal stress. The alkaline phosphatases (PMEase and PDEase) are quite stable enzymes and require a narrow range of pH (pH 10–10.2) and temperature (35–40 °C) for their optimal activity.A pH of 10, 10.2 and 10.2 supported optimal activity of cellular PMEase, cellular PDEase and extracellular PMEase, respectively, whereas temperatures of 35, 38 and 40 °C were required for their optimal activity. The requirement for Ca²⁺ and Mg²⁺ as macronutrients and the significance of the micronutrients Zn²⁺, Co²⁺, Fe²⁺, Mn²⁺ and Cu²⁺ in APase activity in the cyanobacterium suggests nutritional regulation of enzyme activity in A. oryzae. The metals Pb²⁺, Cr⁶⁺ and Ni²⁺ severely inhibited APase activity, whereas the NaCl stress had a dual role, which was concentration dependent. NaCl stress at lower concentrations (≤20 mM) caused an increase in cellular PMEase activity while its higher concentration (>20 mM) favoured release of the extracellular PMEase. The decrease in cellular activity and an increase in extracellular activity suggest that the higher concentrations of salt stimulate the release of the enzyme.The data suggest that the cyanobacterium A. oryzae possess a potential application as biofertilizer in high salinity and alkaline (Ca²⁺-rich) soils because of its ability to release PO₄³⁻ enzymatically under these conditions.     

Purification and biochemical characterization of a new alkali-stable laccase from Trametes sp. isolated in Tunisia: role of the enzyme in olive mill waste water treatment

A white-rot basidiomycete, isolated from decayed acacia wood (from Northwest of Tunisia) and identified as Trametes sp, was selected in a broad plate screening because of its ability to decolorize and dephenolize olive oil mill wastewater (OMW) efficiently. The major laccase was purified and characterized as a monomeric protein with apparent molecular mass of 61 kDa (SDS-PAGE). It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 4.0 and a temperature of 60 °C. The purified laccase is stable at alkaline pH values. The enzyme retained 50 % of its activity after 90 min of incubation at 55 °C. Using ABTS, this laccase presented K _m and V _max values of 0.05 mM and 212.73 μmoL min^?1 mg^?1, respectively. It has shown a degrading activity towards a variety of phenolic compounds. The purified laccase was partially inhibited by Fe²⁺, Zn²⁺, Cd²⁺ and Mn²⁺, while Cu²⁺ acted as inducer. EDTA (10 mM) and NaN₃ (10 mM) were found to completely inhibit its activity. 73 % OMW was dephenolized after 315 min incubation at 30 °C with 2 U mL^?1 of laccase and 2 mM HBT.     

Enzyme promiscuity in natural environments: alkaline phosphatase in the ocean

Alkaline phosphatase (APase) is one of the marine enzymes used by oceanic microbes to obtain inorganic phosphorus (P_i) from dissolved organic phosphorus to overcome P-limitation. Marine APase is generally recognized to perform P-monoesterase activity. Here we integrated a biochemical characterization of a specific APase enzyme, examination of global ocean databases, and field measurements, to study the type and relevance of marine APase promiscuity. We performed an in silico mining of phoA homologs, followed by de novo synthesis and heterologous expression in E. coli of the full-length gene from Alteromonas mediterranea, resulting in a recombinant PhoA. A global analysis using the TARA Oceans, Malaspina and other metagenomic databases confirmed the predicted widespread distribution of the gene encoding the targeted PhoA in all oceanic basins throughout the water column. Kinetic assays with the purified PhoA enzyme revealed that this enzyme exhibits not only the predicted P-monoester activity, but also P-diesterase, P-triesterase and sulfatase activity as a result of a promiscuous behavior. Among all activities, P-monoester bond hydrolysis exhibited the highest catalytic activity of APase despite its lower affinity for phosphate monoesters. APase is highly efficient as a P-monoesterase at high substrate concentrations, whereas promiscuous activities of APase, like diesterase, triesterase, and sulfatase activities are more efficient at low substrate concentrations. Strong similarities were observed between the monoesterase:diesterase ratio of the purified PhoA protein in the laboratory and in natural seawater. Thus, our results reveal enzyme promiscuity of APase playing potentially an important role in the marine phosphorus cycle.Subject terms: Microbial biooceanography, Biogeochemistry, Microbial ecology     

Strontium ranelate stimulates the activity of bone-specific alkaline phosphatase: interaction with Zn2+ and Mg2+

Strontium ranelate (SR) is an orally administered and bone-targeting anti-osteoporotic agent that increases osteoblast-mediated bone formation while decreasing osteoclastic bone resorption, and thus reduces the risk of vertebral and femoral bone fractures in postmenopausal women with osteoporosis. Osteoblastic alkaline phosphatase (ALP) is a key enzyme involved in the process of bone formation and osteoid mineralization. In this study we investigated the direct effect of strontium (SR and SrCl₂) on the activity of ALP obtained from UMR106 osteosarcoma cells, as well as its possible interactions with the divalent cations Zn²⁺ and Mg²⁺. In the presence of Mg²⁺, both SR and SrCl₂ (0.05–0.5 mM) significantly increased ALP activity (15–66 % above basal), and this was dose-dependent in the case of SR. The stimulatory effect of strontium disappeared in the absence of Mg²⁺. The cofactor Zn²⁺ also increased ALP activity (an effect that reached a plateau at 2 mM), and co-incubation of 2 mM Zn²⁺ with 0.05–0.5 mM SR showed an additive effect on ALP activity stimulation. SR induced a dose-dependent decrease in the Km of ALP (and thus an increase in affinity for its substrate) with a maximal effect at 0.1 mM. Co-incubation with 2 mM Zn²⁺ further decreased Km in all cases. These direct effects of SR on osteoblastic ALP activity could be indicating an alternative mechanism by which this compound may regulate bone matrix mineralization.     

Expression, purification and characterization of flavin reductase from Citrobacter freundii A1

Flavin reductase plays an important biological role in catalyzing the reduction of flavin by NAD(P)H oxidation. The gene that codes for flavin reductase from Citrobacter freundii A1 was cloned and expressed in Escherichia coli BL21(DE3)pLysS. In this study, we aimed to characterize the purified recombinant flavin reductase of C. freundii A1. The recombinant enzyme was purified to homogeneity and the biochemical profiles, including the effect of pH, temperature, metal ions and anions on flavin reductase activity and stability, were determined. This enzyme exhibited optimum activity at 45 °C in a 10-min reaction at pH 7.5 and was stable at temperatures up to 30 °C. At 0.1 mM concentration of metal ions, flavin reductase activity was stimulated by divalent cations including Mn²⁺, Sr²⁺, Ni²⁺, Sn²⁺, Ba²⁺, Co²⁺, Mg²⁺, Ca²⁺ and Pb²⁺. Ag⁺ was noticeably the strongest inhibitor of recombinant flavin reductase of C. freundii A1. This enzyme should not be defined as a standard flavoprotein. This is the first attempt to characterize flavin reductase of C. freundii origin.     

The effects of magnesium and calcium ions on phosphatase activities at alkaline pH in the molar region of newborn mice

Summary The hydrolysis of ATP, AMP and glycerophosphate (GP) at alkaline pH in mineralizing bone and teeth of young mice has been studied histochemically. The substrates were visibly hydrolyzed to the same degree in osteoblasts, cells of stratum intermedium, odontoblasts and subodontoblasts at Ca²⁺ concentrations ranging from 10 mM to 600 mM. In the ameloblasts, however, only ATP was hydrolyzed. The ATPase activities gradually decreased at increasing Mg²⁺/Ca²⁺ ratios. The AMPase and GPase activities, on the other hand, were visibly unaffected. Marked cellular staining, including the nuclei was seen with AMP and GP as substrates when only Mg²⁺ ions were added. No ATPase activity at all could be recorded in media containing Mg²⁺ but no Ca²⁺ ions. The different phosphatase activities in cells involved in hard tissue formation were identically affected by preincubations with solutions containing various concentrations of Ca²⁺ or Mg²⁺ ions. The ATPase activity in striated muscle fibres and blood vessel walls, however, was affected differently by the same procedure.The results indicate that the phosphatase activities recorded in osteoblasts, cells of stratum intermedium, odontoblasts and subodontoblasts at alkaline pH belong to one single enzyme. The results also imply that CaATP is the preferred substrate in the enzymatic hydrolysis of ATP in hard-tissue-forming cells.     

A novel malic enzyme gene, <Emphasis Type="Italic">Mime2</Emphasis>, from <Emphasis Type="Italic">Mortierella isabellina</Emphasis> M6-22 contributes to lipid accumulation

Objective

This study was aimed at cloning and characterizing a novel malic enzyme (ME) gene of Mortierella isabellina M6-22 and identifying its relation with lipid accumulation.

Methods

Mime2 was cloned from strain M6-22. Plasmid pET32aMIME2 was constructed to express ME of MIME2 in Escherichia coli BL21. After purification, the optimal pH and temperature of MIME2, as well as K_m and V_max for NADP⁺ were determined. The effects of EDTA or metal ions (Mn²⁺, Mg²⁺, Co²⁺, Cu²⁺, Ca²⁺, or Zn²⁺) on the enzymatic activity of MIME2 were evaluated. Besides, plasmid pRHMIME2 was created to express MIME2 in Rhodosporidium kratochvilovae YM25235, and its cell lipid content was measured by the acid-heating method. The optimal pH and temperature of MIME2 are 5.8 and 30 °C, respectively.

Results

The act ivity of MIME2 was significantly increased by Mg²⁺, Ca²⁺, or Mn²⁺ at 0.5 mM but inhibited by Cu²⁺ or Zn²⁺ (p?<?0.05). The optimal enzymatic activity of MIME2 is 177.46 U/mg, and the K_m and V_max for NADP⁺ are 0.703 mM and 156.25 μg/min, respectively. Besides, Mime2 transformation significantly increased the cell lipid content in strain YM25235 (3.15?±?0.24 vs. 2.17?±?0.31 g/L, p?<?0.01).

Conclusions

The novel ME gene Mime2 isolated from strain M6-22 contributes to lipid accumulation in strain YM25235.

     

Potentiality of Azolla as a suitable P-biofertilizer under salinity through acid phosphatase activity

The cellular and extracellular acid phosphatase (APase, Electrical Conductivity (EC) 3.1.3.2) activities in different Azolla spp. were induced in P-deprived condition after 48 h and maximum was on the 12th day of incubation. APase activity has been investigated at different levels of NaCl. Highest cellular and extracellular APase activities were observed in A. microphylla at 20 (2-fold of the control) and 30 mM (3.2-fold of the control) NaCl, respectively, while lowest were in A. filiculoides. Presence of different concentrations of micro- and macronutrients in saline medium regulated the APase activity of the Azolla. Ca²⁺ played a major role in enhancing the APase activity under salinity. Heavy metals did not affect APase activity at the lower level, whereas its higher level inhibited the activity. The enhancement in the APase activity under moderate salinity suggests the major contribution of APase activity during salt-responsive physiological adaptation of Azolla possibly by providing maximum inorganic phosphate to the association, which might be helpful in compensating for the energy crisis caused by salt-induced inhibition in photosynthetic machinery and also in regulating the nutrient imbalances during salt shock or process of adaptation towards heavily polluted agricultural land.     

Role of Vanadium (V) in the Differentiation of C3H10t1/2 Cells Towards Osteoblast Lineage: A Comparative Analysis with Other Trace Elements

In recent time, vanadium compounds are being used as antidiabetic drug and in orthopedic implants. However, the exact role of this incorporated vanadium in improving the quality of bone structure and morphology is not known. The impact of vanadium ion was studied and compared to other trace metal ions with respect to the proliferation and osteoblast differentiation of C3H10t1/2 cells. Toxicity profile of these trace metal ions revealed a descending toxicity trend of Fe²⁺ > Zn²⁺ > Cu²⁺ > Co²⁺ > Mn²⁺ > V⁵⁺ > Cr²⁺. The effect of vanadium and other trace metal ions on osteoblast differentiation was evaluated by culturing the cells for 10 days in osteoblastic medium supplemented with different trace ions at concentrations lower than their cytotoxic doses. The results indicated that vanadium has maximum impact on the induction of osteoblast differentiation by upregulating alkaline phosphatase activity and mineralization by up to 145 and 150 %, respectively (p?<?0.05), over control. Cu²⁺ and Zn²⁺ had a mild inhibitory effect, while Mn²⁺, Fe²⁺, and Co²⁺ demonstrated a clear decrease in osteoblast differentiation when compared to the control. The data as presented here demonstrate that orthopedic implants, if supplemented with trace metals like vanadium, may provide a source of better model for bone formation and its turnover.     

Cloning of an alkaline phosphatase gene from the moderately thermophilic bacterium<Emphasis Type="Italic"> Meiothermus ruber</Emphasis> and characterization of the recombinant enzyme

A gene that codes for an alkaline phosphatase was cloned from the thermophilic bacterium Meiothermus ruber, and its nucleotide sequence was determined. The deduced amino acid sequence indicates that the enzyme precursor including the putative signal sequence is composed of 503 amino acid residues and has an estimated molecular mass of 54,229 Da. Comparison of the peptide sequence with that of the prototype alkaline phosphatase from Escherichia coli revealed conservation of the regions in the vicinity of the corresponding phosphorylation site and metal binding sites. The protein was expressed in E. coli and its enzymatic properties were characterized. In the absence of exogenously added metal ions, activity was negligible; to obtain maximal activity, addition of free Mg²⁺ ions was required. Zn²⁺ ions had an inhibitory effect on the activity of the M. ruber enzyme. The pH and temperature optima for activity were found to be 11.0 and 62°C, respectively. The enzyme was moderately thermostable: it retained about 50% activity after incubation for 6 h at 60°C, whereas at 80°C it was completely inactivated within 2 h. The Michaelis constant for cleavage of 4-nitrophenylphosphate was 0.055 mM. While having much in common with other alkaline phosphatases, the M. ruber enzyme presents some unique features, such as a very narrow pH range for activity and an absolute requirement for magnesium for activity.Communicated by G. P. Georgiev     

Heterospecific cloning of Arabidopsis thaliana cDNAs by direct complementation of pyrimidine auxotrophic mutants of Saccharomyces cerevisiae. I. Cloning and sequence analysis of two cDNAs catalysing the second,fifth and sixth steps of the de novo pyrimidine biosynthesis pathway

A one-step mutant of Escherichia coli K-12 lacking both glucose-1-phosphatase (Agp) and pH 2.5 acid phosphatase (AppA) activities in the periplasmic space was isolated. The mutation which mapped close to ch1B, at 87 min on the E. coli linkage map, also caused the loss of alkaline phosphatase (PhoA) activity, even when this activity was expressed from TnphoA fusions to genes encoding periplasmic or membrane proteins. A DNA fragment that complements the mutation was cloned and shown to carry the dsbA gene, which encodes a periplasmic disulphide bond-forming factor. The mutant had an ochre triplet in dsbA, truncating the protein at amino acid 70. Introduction of TnphoA fusions into a plasmid-borne dsbA gene resulted in DsbA-PhoA hybrid proteins that were all exported to the periplasmic space in both dsbA ⁺ and dsbA strains. They belong to three different classes, depending on the length of the DsbA fragment fused to PhoA. When PhoA was fused to an amino-terminal DsbA heptapeptide, the protein was only seen in the periplasm of a dsbA ⁺ strain, as in the case of wild-type PhoA. Hybrid proteins missing up to 29 amino acids at the carboxy-terminus of DsbA were stable and retained both the DsbA and PhoA activities. Those with shorter DsbA fragments that still carried the -Cys-ProHis-Cys-motif were rapidly degraded (no DsbA activity). The presence is discussed of a structural domain lying around amino acid 170 of DsbA and which is probably essential for its folding into a proteolytic-resistant and enzymatically active form.     

First Thermostable Endo-β-1,4-Glucanase from Newly Isolated Xanthomonas sp. EC102

A novel gene encoding thermostable endoglucanase was identified in Xanthomonas sp. EC102 from soil. The gene had 1,458 base pairs of open reading frame, which encode a 52-kDa protein of 486 amino acid residues. Sequence of the amino acid residues was similar with the endoglucanase from Xanthomonas campestris pv. campestris ATCC33913 (GenBank Accession No. NP_638867.1) (94 % identity). The endoglucanase was overexpressed in Escherichia coli BL21 and purified. Temperature for the highest enzymatic activity was 70 °C and pH optima was pH 5.5. The specific activity of the endoglucanase toward carboxymethylcellulose (CMC) was approximately 2 μmol min^?1 mg^?1, V _max for CMC was 1.44 μmol mg^?1 min^?1, and K _m values was 25.6 mg mL^?1. The EC102 endoglucanase was stable at temperatures up to 60 °C, and it was activated by 0.1 mM of Mn²⁺ and Co²⁺. This is the first report about thermostable endoglucanase from Xanthomonas sp.     

Characterization of the co-purified invertase and β-glucosidase of a multifunctional extract from Aspergillus terreus

The filamentous fungus Aspergillus terreus secretes both invertase and β-glucosidase when grown under submerged fermentation containing rye flour as the carbon source. The aim of this study was to characterize the co-purified fraction, especially the invertase activity. An invertase and a β-glucosidase were co-purified by two chromatographic steps, and the isolated enzymatic fraction was 139-fold enriched in invertase activity. SDS-PAGE analysis of the co-purified enzymes suggests that the protein fraction with invertase activity was heterodimeric, with subunits of 47 and 27 kDa. Maximal invertase activity, which was determined by response surface methodology, occurred in pH and temperature ranges of 4.0–6.0 and 55–65 °C, respectively. The invertase in co-purified enzymes was stable for 1 h at pH 3.0–10.0 and maintained full activity for up to 1 h at 55 °C when diluted in water. Invertase activity was stimulated by 1 mM concentrations of Mn²⁺ (161 %), Co²⁺ (68 %) and Mg²⁺ (61 %) and was inhibited by Al³⁺, Ag⁺, Fe²⁺ and Fe³⁺. In addition to sucrose, the co-purified enzymes hydrolyzed cellobiose, inulin and raffinose, and the apparent affinities for sucrose and cellobiose were quite similar (K_M = 22 mM). However, in the presence of Mn²⁺, the apparent affinity and V_max for sucrose hydrolysis increased approximately 2- and 2.9-fold, respectively, while for cellobiose, a 2.6-fold increase in V_max was observed, but the apparent affinity decreased 5.5-fold. Thus, it is possible to propose an application of this multifunctional extract containing both invertase and β-glucosidase to degrade plant biomass, thus increasing the concentration of monosaccharides obtained from sucrose and cellobiose.     

Potent inhibition of dinuclear zinc(II) peptidase,an aminopeptidase from <Emphasis Type="Italic">Aeromonas proteolytica</Emphasis>, by 8-quinolinol derivatives: inhibitor design based on Zn<Superscript>2+</Superscript> fluorophores,kinetic, and X-ray crystallographic study

The selective inhibition of an aminopeptidase from Aeromonas proteolytica (AAP), a dinuclear Zn²⁺ hydrolase, by 8-quinolinol (8-hydroxyquinoline, 8-HQ) derivatives is reported. We previously reported on the preparation of 8-HQ-pendant cyclens as Zn²⁺ fluorophores (cyclen is 1,4,7,10-tetraazacyclododecane), in which the nitrogen and phenolate of the 8-HQ units (as well as the four nitrogens of cyclen) bind to Zn²⁺ in a bidentate manner to form very stable Zn²⁺ complexes at neutral pH (K _d = 8–50 fM at pH 7.4). On the basis of this finding, it was hypothesized that 8-HQ derivatives have the potential to function as specific inhibitors of Zn²⁺ enzymes, especially dinuclear Zn²⁺ hydrolases. Assays of 8-HQ derivatives as inhibitors were performed against commercially available dinuclear Zn²⁺ enzymes such as AAP and alkaline phosphatase. 8-HQ and the 5-substituted 8-HQ derivatives were found to be competitive inhibitors of AAP with inhibition constants of 0.16–29 μM at pH 8.0. The nitrogen at the 1-position and the hydroxide at the 8-position of 8-HQ were found to be essential for the inhibition of AAP. Fluorescence titrations of these drugs with AAP and an X-ray crystal structure analysis of an AAP–8-HQ complex (1.3-Å resolution) confirmed that 8-HQ binds to AAP in the “Pyr-out” mode, in which the hydroxide anion of 8-HQ bridges two Zn²⁺ ions (Zn1 and Zn2) in the active site of AAP and the nitrogen atom of 8-HQ coordinates to Zn1 (Protein Data Bank code 3VH9).     

Stimulation of root acid phosphatase by phosphorus deficiency is regulated by ethylene in Medicago falcata

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.     

Cloning, Expression and Characterization of a Trehalose Synthase Gene From Rhodococcus opacus

Trehalose is a unique disaccharide capable of protecting proteins against environmental stress. A novel trehalose synthase (TreS) gene from Rhodococcus opacus was cloned and expressed in Escherichia coli Top10 and BL21 (DE3) pLysS, respectively. The recombinant TreS showed a molecular mass of 79 kDa. Thin layer chromatography (TLC) result suggested that this enzyme had the ability to catalyze the mutual conversion of maltose and trehalose. Moreover, high-performance liquid chromatography (HPLC) result suggested that glucose appeared as a byproduct with a conversion rate of 12 %. The purified recombinant enzyme had an optimum temperature of 25 °C and pH optimum around 7.0. Kinetic analysis revealed that the K _m for trehalose was around 98 mM, which was a little higher than that of maltose. The preferred substrate of TreS was maltose according to the analysis of k _cat/K _m. Both 1 and 10 mM of Hg²⁺, Cu²⁺ and Al³⁺ could inhibit the TreS activity, while only 1 mM of Ca²⁺ and Mn²⁺ could increase its activity. Five amino acid residues, Asp²⁴⁴, Glu²⁸⁶, Asp³⁵⁴, His¹⁴⁷ and His³⁵³, were shown to be conserved in R. opacus TreS, which were also important for α-amylase family enzyme catalysis.