Requirement For Divalent Cations Research Articles

Biochemical characterization of a thermally stable, acidophilic and surfactant-tolerant xylanase from Aspergillus awamori AFE1 and hydrolytic efficiency of its immobilized form

Capital intensiveness of bioconversion of lignocellulosic biomass can be unraveled by producing stable and highly efficient enzyme from phytophilic microbes. The present study revealed biodegradation efficiency of immobilized and free xylanase from Aspergillus awamori AFE1 and the enzyme physicochemical properties. The crude xylanase was subjected to three step purification followed by determination of biochemical properties. The homogenously purified xylanase had an overall 6.86-fold purification and 5.20 % yield coupled with a molecular weight of ~ 48 kDa. Free and immobilized xylanase exhibited optimum activity at (50 °C, pH 4) and (70 °C, pH 5) respectively while thermal and pH stability for free were achieved at a range of 30–90 °C and pH 2–11 respectively. Xylanase was tolerant and enhanced by organic solvents, detergents and inhibitors investigated except ethanol and Cysteine while Mn2+ exceptionally increased xylanase activity and its inhibition by EDTA showed requirement for divalent cations. Km, 0.15 mM and 1 mM and Vmax, 5 × 103 µM/min and 1 × 104 µM/min were obtained for free and immobilized xylanase. Interestingly, immobilized xylanase showed recycling efficiency of five cycles with higher degradation efficiency over solubilized xylanase. Both free and immobilized xylanase novel characteristics showed suitability for industrial and biotechnological applications.

A YoeB toxin cleaves both RNA and DNA

Type II toxin-antitoxin systems contain a toxin protein, which mediates diverse interactions within the bacterial cell when it is not bound by its cognate antitoxin protein. These toxins provide a rich source of evolutionarily-conserved tertiary folds that mediate diverse catalytic reactions. These properties make toxins of interest in biotechnology applications, and studies of the catalytic mechanisms continue to provide surprises. In the current work, our studies on a YoeB family toxin from Agrobacterium tumefaciens have revealed a conserved ribosome-independent non-specific nuclease activity. We have quantified the RNA and DNA cleavage activity, revealing they have essentially equivalent dose-dependence while differing in requirements for divalent cations and pH sensitivity. The DNA cleavage activity is as a nickase for any topology of double-stranded DNA, as well as cleaving single-stranded DNA. AtYoeB is able to bind to double-stranded DNA with mid-micromolar affinity. Comparison of the ribosome-dependent and -independent reactions demonstrates an approximate tenfold efficiency imparted by the ribosome. This demonstrates YoeB toxins can act as non-specific nucleases, cleaving both RNA and DNA, in the absence of being bound within the ribosome.

Molecular characterization of Plasmodium falciparum DNA-3-methyladenine glycosylase

BackgroundThe emergence of artemisinin-resistant malaria parasites highlights the need for novel drugs and their targets. Alkylation of purine bases can hinder DNA replication and if unresolved would eventually result in cell death. DNA-3-methyladenine glycosylase (MAG) is responsible for the repair of those alkylated bases. Plasmodium falciparum (Pf) MAG was characterized for its potential for development as an anti-malarial candidate.MethodsNative PfMAG from crude extract of chloroquine- and pyrimethamine-resistant P. falciparum K1 strain was partially purified using three chromatographic procedures. From bio-informatics analysis, primers were designed for amplification, insertion into pBAD202/D-TOPO and heterologous expression in Escherichia coli of recombinant PfMAG. Functional and biochemical properties of the recombinant enzyme were characterized.ResultsPfMAG activity was most prominent in parasite schizont stages, with a specific activity of 147 U/mg (partially purified) protein. K1 PfMAG contained an insertion of AAT (coding for asparagine) compared to 3D7 strain and 16% similarity to the human enzyme. Recombinant PfMAG (74 kDa) was twice as large as the human enzyme, preferred double-stranded DNA substrate, and demonstrated glycosylase activity over a pH range of 4–9, optimal salt concentration of 100–200 mM NaCl but reduced activity at 250 mM NaCl, no requirement for divalent cations, which were inhibitory in a dose-dependent manner.ConclusionPfMAG activity increased with parasite development being highest in the schizont stages. K1 PfMAG contained an indel AAT (asparagine) not present in 3D7 strain and the recombinant enzyme was twice as large as the human enzyme. Recombinant PfMAG had a wide range of optimal pH activity, and was inhibited at high (250 mM) NaCl concentration as well as by divalent cations. The properties of PfMAG provide basic data that should be of assistance in developing anti-malarials against this potential parasite target.

Biochemical characterization of bacterial FeoBs: A perspective on nucleotide specificity

Iron is an essential requirement for the survival and virulence of most bacteria. The bacterial ferrous iron transporter protein FeoB functions as a major reduced iron transporter in prokaryotes, but its biochemical mechanism has not been fully elucidated. In the present study, we compared enzymatic properties of the cytosolic portions of pathogenic bacterial FeoBs to elucidate each bacterial strain-specific characteristic of the Feo system. We show that bacterial FeoBs are classified into two distinct groups that possess either a sole GTPase or an NTPase with a substrate promiscuity. This difference in nucleotide preference alters cellular requirements for monovalent and divalent cations. While the hydrolytic activity of the GTP-dependent FeoBs was stimulated by potassium, the action of the NTP-dependent FeoBs was not significantly affected by the presence of monovalent cations. Mutation of Asn11, having a role in potassium-dependent GTP hydrolysis, changed nucleotide specificity of the NTP-dependent FeoB, resulting in loss of ATPase activity. Sequence analysis suggested a possible association of alanine in the G5 motif for the NTP-dependent activity in FeoBs. This demonstration of the distinct enzymatic properties of bacterial FeoBs provides important insights into mechanistic details of Feo iron transport processes, as well as offers a promising species-specific anti-virulence target.

Characterization of an intertidal zone metagenome oligoribonuclease and the role of the intermolecular disulfide bond for homodimer formation and nuclease activity

The gene encoding MG Orn has been identified from a metagenomic library created from the intertidal zone in Svalbard and encodes a protein of 184 amino acid residues. The mg orn gene has been cloned, recombinantly expressed in Escherichia coli, and purified to homogeneity. Biochemical characterization of the enzyme showed that it efficiently degrades short RNA oligonucleotide substrates of 2mer to 10mer of length and has an absolute requirement for divalent cations for optimal activity. The enzyme is more heat‐labile than its counterpart from E. coli and exists as a homodimer in solution. The crystal structure of the enzyme has been determined to a resolution of 3.15 Å, indicating an important role of a disulfide bridge for the homodimer formation and as such for the function of MG Orn. Substitution of the Cys110 residue with either Gly or Ala hampered the dimer formation and severely affected the enzyme's ability to act on RNA. A conserved loop containing His128‐Tyr129‐Arg130 in the neighboring monomer is probably involved in efficient binding and processing of longer RNA substrates than diribonucleotides.

Apyrases in Arabidopsis thaliana

Apyrases belong to the ATPase family of enzymes that hydrolyze phosphoanhydride bonds of nucleoside tri- and di-phosphates. These enzymes differ markedly from other phosphohydrolases due to their high specific activity, broad divalent cation requirement, broad nucleotide substrate specificity, and insensitivity to various inhibitors. In the past 30Â years, apyrases have been frequently studied in mammals. In comparison, research of apyrases in plants has received little attention, despite the growth of plants being closely related to the apyrases. In this review, we summarize the research of the apyrases in Arabidopsis thaliana and point to the possible future directions of research. Apyrases have seven members found in Arabidopsis thaliana, each with different properties and functions. Currently, the characterization and functions of AtAPY1 and AtAPY2 have been reported, though, to the best of our knowledge, the other apyrase members (AtAPY3 to 7) have not yet been sufficiently described. In this review, we also summarize the progress being made and the difficulties encountered in apyrase research in Arabidopsis thaliana.

Characterization of a nontypeable Haemophilus influenzae thermonuclease.

Nontypeable Haemophilus influenzae (NTHi) has been shown to form biofilms, comprised of extracellular DNA (eDNA), in the middle ear and bronchus during clinical infections. Studies in our laboratory have shown that NTHi possesses a homolog of Staphylococcus aureus thermonuclease (staphylococcal thermonuclease), NTHi nuclease (NTHi Nuc, HI_1296). This enzyme had similar size, heat stability, and divalent cation requirements to those of the staphylococcal homolog as determined by light scattering and circular dichroism spectroscopy. Small angle X-ray scattering (SAXS) analysis suggested an overall shape and substrate-binding site comparable to those of staphylococcal nuclease. However, NTHi Nuc was approximately 25-fold more active in fluorescence resonance energy transfer (FRET) activity assay than staphylococcal thermonuclease. Homology modeling implicates shorter NTHi Nuc loops near the active site for this enhanced activity.

Identification and characterization of a plastidial phosphatidylglycerophosphate phosphatase in Arabidopsis thaliana.

Phosphatidylglycerol (PG) is the only phospholipid in the thylakoid membranes of chloroplasts of plants, and it is also found in extraplastidial membranes including mitochondria and the endoplasmic reticulum. Previous studies showed that lack of PG in the pgp1-2 mutant of Arabidopsis deficient in phosphatidylglycerophosphate (PGP) synthase strongly affects thylakoid biogenesis and photosynthetic activity. In the present study, the gene encoding the enzyme for the second step of PG synthesis, PGP phosphatase, was isolated based on sequence similarity to the yeast GEP4 and Chlamydomonas PGPP1 genes. The Arabidopsis AtPGPP1 protein localizes to chloroplasts and harbors PGP phosphatase activity with alkaline pH optimum and divalent cation requirement. Arabidopsis pgpp1-1 mutant plants contain reduced amounts of chlorophyll, but photosynthetic quantum yield remains unchanged. The absolute content of plastidial PG (34:4; total number of acyl carbons:number of double bonds) is reduced by about 1/3, demonstrating that AtPGPP1 is involved in the synthesis of plastidial PG. PGP 34:3, PGP 34:2 and PGP 34:1 lacking 16:1 accumulate in pgpp1-1, indicating that the desaturation of 16:0 to 16:1 by the FAD4 desaturase in the chloroplasts only occurs after PGP dephosphorylation.

Calcium-dependent generation of N-acylethanolamines and lysophosphatidic acids by glycerophosphodiesterase GDE7

N-Acylethanolamines form a class of lipid mediators and include an endocannabinoid arachidonoylethanolamide (anandamide), analgesic and anti-inflammatory palmitoylethanolamide, and appetite-suppressing oleoylethanolamide. In animal tissues, N-acylethanolamines are synthesized from N-acylated ethanolamine phospholipids directly by N-acylphosphatidylethanolamine-hydrolyzing phospholipase D or through multi-step pathways via N-acylethanolamine lysophospholipids. We previously reported that glycerophosphodiesterase (GDE) 4, a member of the GDE family, has lysophospholipase D (lysoPLD) activity hydrolyzing N-acylethanolamine lysophospholipids to N-acylethanolamines. Recently, GDE7 was shown to have lysoPLD activity toward lysophosphatidylcholine to produce lysophosphatidic acid (LPA). Here, we examined the reactivity of GDE7 with N-acylethanolamine lysophospholipids as well as the requirement of divalent cations for its catalytic activity. When overexpressed in HEK293 cells, recombinant GDE7 proteins of human and mouse showed lysoPLD activity toward N-palmitoyl, N-oleoyl, and N-arachidonoyl-lysophosphatidylethanolamines and N-palmitoyl-lysoplasmenylethanolamine to generate their corresponding N-acylethanolamines and LPAs. However, GDE7 hardly hydrolyzed glycerophospho-N-palmitoylethanolamine. Overexpression of GDE7 in HEK293 cells increased endogenous levels of N-acylethanolamines and LPAs. Interestingly, GDE7 was stimulated by micromolar concentrations of Ca2+ but not by millimolar concentrations of Mg2+, while GDE4 was stimulated by Mg2+ but was insensitive to Ca2+. GDE7 was widely distributed in various tissues of humans and mice with the highest levels in their kidney tissues. These results suggested that GDE7 is a novel Ca2+-dependent lysoPLD, which is involved in the generation of both N-acylethanolamines and LPAs.

Concerted action of NIC relaxase and auxiliary protein MobC in RA3 plasmid conjugation.

Conjugative transfer of the broad-host-range RA3 plasmid, the archetype of the IncU group, relies on the relaxase NIC that belongs to the as yet uncharacterized MOBP4 subfamily. NIC contains the signature motifs of HUH relaxases involved in Tyr nucleophilic attack. However, it differs in the residue involved in His activation for cation coordination and was shown here to have altered divalent cation requirements. NIC is encoded in the mobC-nic operon preceded directly by oriT, where mobC encodes an auxiliary transfer protein with a dual function: autorepressor and stimulator of conjugative transfer. Here an interplay between MobC and NIC was demonstrated. MobC is required for efficient NIC cleavage of oriT in supercoiled DNA whereas NIC assists MobC in repression of the mobC-nic operon. A 7-bp arm of IR3 (IR3a) was identified as the binding site for NIC and the crucial nucleotides in IR3a for NIC recognition were defined. Fully active oriTRA3 was delineated to a 47-bp DNA segment encompassing a conserved cleavage site sequence, the NIC binding site IR3a and the MobC binding site OM . This highly efficient RA3 conjugative system with defined requirements for minimal oriT could find ample applications in biotechnology and computational biology where simple conjugative systems are needed.

Purification and characterization of a mycelial mucin specific lectin from Aspergillus panamensis with potent mitogenic and antibacterial activity

A lectin was isolated from Aspergillus panamensis mycelia using single-step affinity chromatography on porcine stomach mucin coupled Sepharose 4B. A purification fold of 19.2 with 62.5% yield was achieved by this method. Gel filtration chromatography showed the native molecular mass of 78kDa and the subunit molecular mass of 39kDa was confirmed by 10% SDS-PAGE and MALDI-TOF analysis. Isoelectric focussing of native A. panamensis lectin revealed it to be an acidic protein with pI 4.5. Carbohydrate content of purified lectin was estimated to be 4.95%. All database search of N-terminal amino acid sequences revealed no homology of the purified lectin with previously reported microbial, viral and animal lectins except 29kDa subunit of heterodimeric Vigna sesquipedalis lectin. The lectin from this fungal source is a homodimer. Lectin was fairly thermostable with pH optima of 6.5–8.0 and had no requirement of divalent metal cations for its activity. Maximum mitogenic activity toward mouse spleen cells was exhibited at 100μg/mL concentration of purified lectin. Results of the antimicrobial assay showed that the lectin had potent antibacterial activity against Bacillus cereus, Staphylococcous aureus, Pseudomonas aeruginosa and Escherichia coli.

Role of monovalent and divalent metal cations in human ribokinase catalysis and regulation.

Human ribokinase (RK) is a member of the ribokinase family, and is the first enzyme responsible for D-ribose metabolism, since D-ribose must first be converted into D-ribose-5-phosphate to be further metabolized and incorporated into ATP or other high energy phosphorylated compounds. Despite its biological importance, RK is poorly characterized in eukaryotes and especially in human. We have conducted a comprehensive study involving catalytic and regulatory features of the human enzyme, focusing on divalent and monovalent metal regulatory effects. Mg(2+), Mn(2+), and Co(2+) support enzyme activity although at different rates, with Mn(2+) being the most effective. Analysis of the divalent cation requirement in the wild type enzyme demonstrates that in addition to that chelated by the nucleotide substrate, an activating cation (either Mn(2+) or Mg(2+)) is required to obtain full activity of the enzyme, with the affinity for both divalent cations being almost the same (4 and 8 µM respectively). Besides metal cation activation, inhibition of the enzyme activity by increasing concentrations of Mn(2+) but not Mg(2+) is observed. Also the role of residues N199 and E202 of the highly conserved NXXE motif present at the active site has been evaluated regarding Mg(2+) and phosphate binding. K(+) (but not Na(+)) and PO4 (3-) activate the wild type enzyme, whereas the N199L and E202L mutants display a dramatic decrease in kcat and require higher free Mg(2+) concentrations than the wild type enzyme to reach maximal activity, and the activating effect of PO4 (3-) is lost. The results demonstrated a complex regulation of the human ribokinase activity where residues Asn199 and Glu202 play an important role.

Purification and Characterization of a Polyextremophilic α -Amylase from an Obligate Halophilic Aspergillus penicillioides Isolate and Its Potential for Souse with Detergents.

An extracellular α-amylase from the obligate halophilic Aspergillus penicillioides TISTR3639 strain was produced and enriched to apparent homogeneity by ammonium sulfate precipitation and Sephadex G100 gel filtration column chromatography. The mass of the purified amylase was estimated to be 42 kDa by SDS-PAGE. With soluble starch as the substrate it had a specific activity of 118.42 U·mg−1 and V max⁡ and K m values of 1.05 µmol·min−1·mg−1 and 5.41 mg·mL−1, respectively. The enzyme was found to have certain polyextremophilic characteristics, with an optimum activity at pH 9, 80°C, and 300 g·L−1 NaCl. The addition of CaCl2 at 2 mM was found to slightly enhance the amylase activity, while ZnCl2, FeCl2, or EDTA at 2 mM was strongly or moderately inhibitory, respectively, suggesting the requirement for a (non-Fe2+ or Zn2+) divalent cation. The enzyme retained more than 80% of its activity when incubated with three different laundry detergents and had a better performance compared to a commercial amylase and three detergents in the presence of increasing NaCl concentrations up to 300 g·L−1. Accordingly, it has a good potential for use as an α-amylase in a low water activity (high salt concentration) and at high pH and temperatures.

PPX1 gene overexpression has no influence on polyphosphates in Saccharomyces cerevisiae.

The role of exopolyphosphatase PPX1 in polyphosphate metabolism in yeasts has been studied in strains of Saccharomyces cerevisiae with inactivated PPX1 and PPN1 genes transformed by the expression vector carrying the yeast PPX1 gene. Exopolyphosphatase activity in transformant strains increased 90- and 40-fold compared to the ΔPPX1 and ΔPPN1 strains, respectively. The purified recombinant exopolyphosphatase PPX1 was similar to the PPX1 of wild strains in its substrate specificity and requirement for divalent metal cations. It was more active with tripolyphosphate and low molecular mass polyphosphates than with high molecular mass polyphosphates and required Mg2+ for its activity. The high level of recombinant PPX1 expression caused no decrease in polyphosphate content in the cells of the transformant. This fact suggests the restricted role of PPX1 in polyphosphate metabolism in yeasts.

Two exopolyphosphatases with distinct molecular architectures and substrate specificities from the thermophilic green-sulfur bacterium Chlorobium tepidum TLS.

The genome of the thermophilic green-sulfur bacterium Chlorobium tepidum TLS possesses two genes encoding putative exopolyphosphatases (PPX; EC 3.6.1.11), namely CT0099 (ppx1, 993 bp) and CT1713 (ppx2, 1557 bp). The predicted polypeptides of 330 and 518 aa residues are Ppx-GppA phosphatases of different domain architectures - the largest one has an extra C-terminal HD domain - which may represent ancient paralogues. Both ppx genes were cloned and overexpressed in Escherichia coli BL21(DE3). While CtPPX1 was validated as a monomeric enzyme, CtPPX2 was found to be a homodimer. Both PPX homologues were functional, K(+)-stimulated phosphohydrolases, with an absolute requirement for divalent metal cations and a marked preference for Mg(2+). Nevertheless, they exhibited remarkably different catalytic specificities with regard to substrate classes and chain lengths. Even though both enzymes were able to hydrolyse the medium-size polyphosphate (polyP) P13-18 (polyP mix with mean chain length of 13-18 phosphate residues), CtPPX1 clearly reached its highest catalytic efficiency with tripolyphosphate and showed substantial nucleoside triphosphatase (NTPase) activity, while CtPPX2 preferred long-chain polyPs (>300 Pi residues) and did not show any detectable NTPase activity. These catalytic features, taken together with the distinct domain architectures and molecular phylogenies, indicate that the two PPX homologues of Chl. tepidum belong to different Ppx-GppA phosphatase subfamilies that should play specific biochemical roles in nucleotide and polyP metabolisms. In addition, these results provide an example of the remarkable functional plasticity of the Ppx-GppA phosphatases, a family of proteins with relatively simple structures that are widely distributed in the microbial world.

Homogeneous purification and characterization of LePGT1 – a membrane‐bound aromatic substrate prenyltransferase involved in secondary metabolism of Lithospermum erythrorhizon

Membrane-bound type prenyltransferases for aromatic substrates play crucial roles in the biosynthesis of various natural compounds. Lithospermumerythrorhizon p-hydroxybenzoate: geranyltransferase (LePGT1), which contains multiple transmembrane α-helices, is involved in the biosynthesis of a red naphthoquinone pigment, shikonin. Taking LePGT1 as a model membrane-bound aromatic substrate prenyltransferase, we utilized a baculovirus-Sf9 expression system to generate a high yield LePGT1 polypeptide, reaching ~1000-fold higher expression level compared with a yeast expression system. Efficient solubilization procedures and biochemical purification methods were developed to extract LePGT1 from the membrane fraction of Sf9 cells. As a result, 80μg of LePGT1 was purified from 150mL culture to almost homogeneity as judged by SDS/PAGE. Using purified LePGT1, enzymatic characterization, e.g. substrate specificity, divalent cation requirement and kinetic analysis, was done. In addition, inhibition experiments revealed that aromatic compounds having two phenolic hydroxyl groups effectively inhibited LePGT1 enzyme activity, suggesting a novel recognition mechanism for aromatic substrates. As the first example of solubilization and purification of this membrane-bound protein family, the methods established in this study will provide valuable information for the precise biochemical characterization of aromatic prenyltransferases as well as for crystallographic analysis of this novel enzyme family.

Characterization of LlaKI, a New Metal Ion-Independent Restriction Endonuclease from Lactococcus lactis KLDS4.

Requirement of divalent cations for DNA cleavage is a general feature of type II restriction enzymes with the exception of few members of this group. A new type II restriction endonuclease has been partially purified from Lactococcus lactis KLDS4. The enzyme was denoted as LlaKI and showed to recognize and cleave the same site as FokI. The enzyme displayed a denatured molecular weight of 50 kDa and behaved as a dimer in solution as evidenced by the size exclusion chromatography. To investigate the role of divalent cations in DNA cleavage by LlaKI, digestion reactions were carried out at different Mg2+, Mn2+, and Ca2+ concentrations. Unlike most of type II restriction endonucleases, LlaKI did not require divalent metal ions to cleave DNA and is one of the few metal-independent restriction endonucleases found in bacteria. The enzyme showed near-maximal levels of activity in 10 mM Tris-HCl pH 7.9, 50 mM NaCl, 10 mM MgCl2, and 1 mM dithiothreitol at 30°C. The presence of DNA modification was also determined and was correlated with the correspondent restriction enzyme.

Characterization of Coumarin-Specific Prenyltransferase Activities inCitrus limonPeel

Coumarins, a large group of polyphenols, play important roles in the defense mechanisms of plants, and they also exhibit various biological activities beneficial to human health, often enhanced by prenylation. Despite the high abundance of prenylated coumarins in citrus fruits, there has been no report on coumarin-specific prenyltransferase activity in citrus. In this study, we detected both O- and C-prenyltransferase activities of coumarin substrates in a microsome fraction prepared from lemon (Citrus limon) peel, where large amounts of prenylated coumarins accumulate. Bergaptol was the most preferred substrate out of various coumarin derivatives tested, and geranyl diphosphate (GPP) was accepted exclusively as prenyl donor substrate. Further enzymatic characterization of bergaptol 5-O-geranyltransferase activity revealed its unique properties: apparent K(m) values for GPP (9 µM) and bergaptol (140 µM) and a broad divalent cation requirement. These findings provide information towards the discovery of a yet unidentified coumarin-specific prenyltransferase gene.

Molecular basis for the distinct divalent cation requirement in the uridylylation of the signal transduction proteins GlnJ and GlnB from Rhodospirillum rubrum

BackgroundPII proteins have a fundamental role in the control of nitrogen metabolism in bacteria, through interactions with different PII targets, controlled by metabolite binding and post-translational modification, uridylylation in most organisms. In the photosynthetic bacterium Rhodospirillum rubrum, the PII proteins GlnB and GlnJ were shown, in spite of their high degree of similarity, to have different requirements for post-translational uridylylation, with respect to the divalent cations, Mg2+ and Mn2+.ResultsGiven the importance of uridylylation in the functional interactions of PII proteins, we have hypothesized that the difference in the divalent cation requirement for the uridylylation is related to efficient binding of Mg/Mn-ATP to the PII proteins. We concluded that the amino acids at positions 42 and 85 in GlnJ and GlnB (in the vicinity of the ATP binding site) influence the divalent cation requirement for uridylylation catalyzed by GlnD.ConclusionsEfficient binding of Mg/Mn-ATP to the PII proteins is required for uridylylation by GlnD. Our results show that by simply exchanging two amino acid residues, we could modulate the divalent cation requirement in the uridylylation of GlnJ and GlnB.Considering that post-translational uridylylation of PII proteins modulates their signaling properties, a different requirement for divalent cations in the modification of GlnB and GlnJ adds an extra regulatory layer to the already intricate control of PII function.

Biochemical characterization and evaluation of cytotoxicity of antistaphylococcal chimeric protein P128.

BackgroundAntibiotic resistant S. aureus infection is a global threat. Newer approaches are required to control this organism in the current scenario. Cell wall degrading enzymes have been proposed as antibacterial agents for human therapy. P128 is a novel antistaphylococcal chimeric protein under development against S. aureus for human use which derives its bacterial cell wall degrading catalytic endopeptidase domain from ORF56, the Phage K tail-structure associated enzyme. Lead therapeutic entities have to be extensively characterized before they are assessed in animals for preclinical safety and toxicity. P128 is effective against antibiotic resistant strains as well as against a panel of isolates of global significance. Its efficacy against S. aureus in vivo has been established in our lab. Against this background, this study describes the characterization of this protein for its biochemical properties and other attributes.ResultsWe evaluated the requirement or effect of divalent cations and the metal ion chelator, EDTA upon biological activity of P128. As the protein is intended for therapeutic use, we tested its activity in presence of body fluids and antibodies specific to P128. For the same reason, we used standard human cell lines to evaluate cytotoxic effects, if any.The divalent cations, calcium and magnesium at upto 25 mM and Zinc upto 2.5 mM neither inhibited nor enhanced P128 activity. Incubation of this protein with EDTA, human serum, plasma and blood also did not alter the antibacterial properties of the molecule. No inhibitory effect was observed in presence of hyper-immune sera raised against the protein. Finally, P128 did not show any cytotoxic effect on HEp2 and Vero cells at the highest concentration (5 mg/mL) tested.ConclusionsThe results presented here throw light on several properties of protein P128. Taken together, these substantiate the potential of P128 for therapeutic use against S. aureus. Further development of the protein and conduct of preclinical safety studies in animals is warranted.