Phosphate Elimination Research Articles

Mechanistic Investigations of the Dehydration Reaction of Lacticin 481 Synthetase Using Site-Directed Mutagenesis

Lantibiotic synthetases catalyze the dehydration of Ser and Thr residues in their peptide substrates to dehydroalanine (Dha) and dehydrobutyrine (Dhb), respectively, followed by the conjugate addition of Cys residues to the Dha and Dhb residues to generate the thioether cross-links lanthionine and methyllanthionine, respectively. In this study ten conserved residues were mutated in the dehydratase domain of the best characterized family member, lacticin 481 synthetase (LctM). Mutation of His244 and Tyr408 did not affect dehydration activity with the LctA substrate whereas mutation of Asn247, Glu261, and Glu446 considerably slowed down dehydration and resulted in incomplete conversion. Mutation of Lys159 slowed down both steps of the net dehydration: phosphorylation of Ser/Thr residues and the subsequent phosphate elimination step to form the dehydro amino acids. Mutation of Arg399 to Met or Leu resulted in mutants that had phosphorylation activity but displayed greatly decreased phosphate elimination activity. The Arg399Lys mutant retained both activities, however. Similarly, the Thr405Ala mutant phosphorylated the LctA substrate but had compromised elimination activity. Finally, mutation of Asp242 or Asp259 to Asn led to mutant enzymes that lacked detectable dehydration activity. Whereas the Asp242Asn mutant retained phosphate elimination activity, the Asp259Asn mutant was not able to eliminate phosphate from a phosphorylated substrate peptide. A model is presented that accounts for the observed phenotypes of these mutant enzymes.

A computational approach to explore <italic>Plasmodium falciparum</italic> 3D7 Chorismate Synthase

Malaria remains the leading cause of deaths attributable to a communicable disease globally, with an unprecedented death toll of over 2 million per year. The reemergence of drug-resistant Plasmodium falciparum, the most fatal human malarial parasite, has focused attention on Shikimate pathway, as it may provide the urgently required novel drug targets owing to lack of homologs of enzymes involved in the this pathway in humans. Chorismate Synthase, the key enzyme of Shikimate pathway, catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate to Chorismate via a 1, 4-trans elimination of phosphate. The strict requirement for a reduced flavin mononucleotide and the anti 1, 4 elimination are both unusual aspects which make CS reaction unique among flavin-dependent enzymes, representing an important target for the chemotherapeutic agents development. The protein sequence of Chorismate Synthase containing 527 residues was obtained from NCBI. The enzyme was characterized using in silico tools. The secondary and tertiary (3D) structure of the enzyme was predicted using CS from other species and validated by various structural quality validation tools. Results of the current study will provide a deep insight about the structure and function of Chorismate Synthase and aid in rational drug designing.

Role of glutamate 243 in the active site of 2-deoxy- scyllo-inosose synthase from Bacillus circulans

2-Deoxy- scyllo-inosose (DOI) synthase is involved in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics and catalyzes the carbocyclic formation from d-glucose-6-phosphate (G-6-P) into DOI. The reaction mechanism is proposed to be similar to that of dehydroquinate (DHQ) synthase in the shikimate pathway, and includes oxidation of C-4, β-elimination of phosphate, reduction of C-4, ring opening, and intramolecular aldol cyclization. To investigate the reaction mechanism of DOI synthase, site-directed mutational analysis of three presumable catalytically important amino acids of DOI synthase derived from the butirosin producer Bacillus circulans (BtrC) was carried out. Steady state and pre-steady state kinetic analysis suggested that E243 of BtrC is catalytically involved in the phosphate elimination step. Further analysis of the mutant E243Q of BtrC using substrate analogue, glucose-6-phosphonate, clearly confirmed that E243 was responsible to abstract a proton at C-5 in G-6-P and set off phosphate elimination. This glutamate residue is completely conserved in all DOI synthases identified so far and the corresponding amino acid of DHQ synthase is completely conserved as asparagine. Therefore, this characteristic glutamate residue of DOI synthase is a key determinant to distinguish the reaction mechanism between DOI synthase and DHQ synthase as well as primary sequence.

Methylglyoxal Is an Intermediate in the Biosynthesis of 6-Deoxy-5-ketofructose-1-phosphate: A Precursor for Aromatic Amino Acid Biosynthesis in Methanocaldococcus jannaschii

A biosynthetic pathway is proposed for creating 6-deoxy-5-ketofructose-1-phosphate (DKFP), a precursor sugar for aromatic amino acid biosynthesis in Methanocaldococcus jannaschii. First, two possible routes were investigated to determine if a modified, established biosynthetic pathway could be responsible for generating 6-deoxyhexoses in M. jannaschii. Both the nucleoside diphosphate mannose pathway and a pathway involving nucleoside diphosphate derivatives of fructose-1-P, fructose-2-P, or fructose-1,6-bisP were tested and eliminated. The established pathways did not produce the expected intermediates nor did the anticipated enzymes have the predicted enzymatic activities. Because neither anticipated pathway could produce DKFP, M. jannaschii glucose-6-P metabolism was studied in detail to establish exactly how glucose-6-P is converted into DKFP. This detailed analysis showed that methylglyoxal and a fructose-1-P- or fructose-1,6-bisP-derived dihydroxyacetone-P fragment are key intermediates in DKFP production. Glucose-6-P readily converts to fructose-6-P, which in turn converts to fructose-1,6-bisP. Fructose-6-P and fructose-1,6-bisP convert into glyceraldehyde-3-P (Ga-P-3), which converts into methylglyoxal by a 2,3-elimination of phosphate. The MJ1585-derived enzyme catalyzes the condensation of methylglyoxal with a dihydroxyacetone-P fragment, which is derived from fructose-1-P and/or fructose-1,6-bisP, generating DKFP. The elimination of phosphate from Ga-P-3 proceeds by both enzymatic and chemical routes in cell extracts, producing sufficient concentrations of methylglyoxal to support the reaction. This work is the first report of methylglyoxal functioning in central metabolism.

Tagging of Phosphopeptides by Phosphate Elimination and Michael Addition: The Effect of Nearest Neighbors on the Reaction Kinetics

Tagging of Phosphopeptides by Phosphate Elimination and Michael Addition: The Effect of Nearest Neighbors on the Reaction Kinetics

Regioselective Synthesis of 4-Alkylpyridines from Pyridine and Aldehydes via Dipole Reversal Process of 1,4-Dihydropyridine Phosphonate

4-Alkylation of pyridine has been accomplished by the reaction of ylides, derivated from 1,4-dihydropyridine phosphonate via phosphonioalkoxy-carbonylation of pyridine with aldehydes and subsequent elimination of diisopropyl phosphate followed by aromatization with potassium tert-butoxide.

Computational intelligence-based optimisation of wastewater treatment plants

Methods of computational intelligence (CI), especially fuzzy control and neuronal networks, are used for controlling and optimising of wastewater treatment plants. Areas of application are the control of sludge water dosage, of phosphate elimination by optimal precipitant dosage as well as an optimal aeration in the nitrification zone. In two municipal wastewater treatment plants with 60,000 and 12,600 person equivalents the controllers have been installed and optimised and they have been in operation for several years. Results of operation of the plants are presented in comparison to previously used classical control. Performance increased significantly and the outflow values could be kept securely below the government requirements without increase of the energy consumption. Peak loads in the inflow were eliminated in the plant and did not increase outflow concentrations. Results of operation for more than three years clearly show that the CI controller is a cost-efficient method for a sustainable rise of performance in municipal wastewater treatment plants.

Lacticin 481 Synthetase Phosphorylates its Substrate during Lantibiotic Production

Lacticin 481 synthetase (LctM) catalyzes the ATP-dependent conversion of a ribosomally synthesized peptide to a polycyclic thioether antibiotic. It is a bifunctional enzyme that dehydrates four Ser/Thr residues to the corresponding dehydro amino acids and catalyzes the conjugate addition of Cys residues to these dehydro residues in a regio- and stereoselective process. We show here that incubation of truncated substrates with LctM results in products that are phosphorylated in the region of dehydration. Furthermore, synthetic peptides containing phosphorylated Ser and/or Thr residues are accepted by the enzyme as substrates resulting in the elimination of phosphate and dehydro amino acid production. This activity is only observed if ADP is added as cosubstrate. These results argue strongly that the enzyme utilizes ATP to phosphorylate the Ser/Thr residues that are targeted for dehydration. ATP does not appear to be required for peptide translocation or cyclization.

Crystal Structure of the NAD Biosynthetic Enzyme Quinolinate Synthase

A gene encoding a quinolinate synthase has been identified in the hyperthermophilic archaeon Pyrococcus horikoshii via genome sequencing. The gene was overexpressed in Escherichia coli, and the crystal structure of the produced enzyme was determined to 2.0 A resolution in the presence of malate, a substrate analogue. The overall structure exhibits a unique triangular architecture composed of a 3-fold repeat of three-layer (alphabetaalpha) sandwich folding. Although some aspects of the fold homologous to the each domain have been observed previously, the overall structure of quinolinate synthase shows no similarity to any known protein structure. The three analogous domains are related to a pseudo-3-fold symmetry. The active site is located at the interface of the three domains and is centered on the pseudo-3-fold axis. The malate molecule is tightly held near the bottom of the active site cavity. The model of the catalytic state during the first condensation step of the quinolinate synthase reaction indicates that the elimination of inorganic phosphate from dihydroxyacetone phosphate may precede the condensation reaction.

Identification of Phosphopeptides by MALDI Q-TOF MS in Positive and Negative Ion Modes after Methyl Esterification

We have developed an efficient, sensitive, and specific method for the detection of phosphopeptides present in peptide mixtures by MALDI Q-TOF mass spectrometry. Use of the MALDI Q-TOF enables selection of phosphopeptides and characterization by CID of the phosphopeptides performed on the same sample spot. However, this type of experiment has been limited by low ionization efficiency of phosphopeptides in positive ion mode while selecting precursor ions of phosphopeptides. Our method entails neutralizing negative charges on acidic groups of nonphosphorylated peptides by methyl esterification before mass spectrometry in positive and negative ion modes. Methyl esterification significantly increases the relative signal intensity generated by phosphopeptides in negative ion mode compared with positive ion mode and greatly increases selectivity for phosphopeptides by suppressing the signal intensity generated by acidic peptides in negative ion mode. We used the method to identify 12 phosphopeptides containing 22 phosphorylation sites from low femtomolar amounts of a tryptic digest of beta-casein and alpha-s-casein. We also identified 10 phosphopeptides containing five phosphorylation sites from an in-gel tryptic digest of 100 fmol of an in vitro autophosphorylated fibroblast growth factor receptor kinase domain and an additional phosphopeptide containing another phosphorylation site when 500 fmol of the digest was examined. The results demonstrate that the method is a fast, robust, and sensitive means of characterizing phosphopeptides present in low abundance mixtures of phosphorylated and nonphosphorylated peptides.

Analysis of Methylglyoxal Metabolism in CHO Cells Grown in Culture.

Recent evidence suggests that several unknown or ill-characterized factors strongly influence cell growth and function in culture. Isolating these factors is necessary in order to maximize culture productivities. Methylglyoxal (MG), a potent protein and nucleic acid modifying agent, has been identified as a player in the signaling pathways associated with cell death and is known to be detrimental to cultured cells. This compound is produced in all mammalian systems by spontaneous phosphate elimination from glycolytic pathway intermediates. A kinetic model that qualitatively describes the cellular distribution of protein-associated MG in the absence of enzymatic adduct formation predicted far lower levels of reversibly bound MG than measured in cultured CHO cells. This suggests that the targeted modification of proteins through enzymatically mediated mechanisms is a significant sink for cellular methylglyoxal. The model was validated with measurements of carbon flux through the glyoxalase pathway to d-lactic acid, a unique end product of MG metabolism in mammalian systems. Fluxes to d-lactic acid of up to 16.8 mmol ml-packed cells(-1) day(-1) were measured with CHO cells grown in batch culture or 100-fold more than found in normal tissues.

Phosphate Elimination and Recovery From by Active Filtration Using Crushed Gas Concrete

Laboratory long-term upstream fixed-bed experiments were carried out to investigate the efficiency of phosphorus elimination with the effluent of a biological sewage treatment plant using crushed gas concrete. The development of the pHvalue in the column outflow as well as the reaction kinetics was investigated. Furthermore, the phosphorus yield was balanced for phosphorus recovery and the calcium phosphate compounds generated were specified by mineralogical analysis methods. These activities were followed by a study with respect to the suitability of the material as raw material for the phosphate industry.

Glycerol and Methylglyoxal Metabolism.

The metabolic connection between glycerol and methylglyoxal (MG) is principally that DHAP, which is an intermediate in the aerobic breakdown of glycerol, is also the major precursor of MG, being the substrate for methylglyoxal synthase (MGS). The synthesis of MG is a consequence of unbalanced metabolism related either to a limitation for phosphate or to excessive carbon flux through the pathways that have the capacity to generate significant pools of DHAP. Cells producing MG produce a poison as an intermediate strategy for survival of metabolic imbalance. Indeed the panoply of metabolic regulation in this sector of catabolism can be seen as a strategy to avoid death by self-poisoning. Glycerol entry into Escherichia coli and Salmonella enterica serovar Typhimurium is facilitated by the aquaglyceroporin, GlpF. A homologous protein in serovar Typhimurium, PduF, facilitates the entry of 1,2-propanediol (Ppd) and is part of the Ppd metabolic pathway. MGS catalyzes the elimination of phosphate from DHAP, forming an enzyme-bound enediol(ate) intermediate that is released from the enzyme, followed by release of inorganic phosphate. The enzyme is highly specific for DHAP. Multiple MG detoxification pathways are found in both E. coli and serovar Typhimurium, but the dominant pathway is the GSH-dependent glyoxalase III system. The KefB and KefC systems have evolved to provide protection during detoxification of electrophiles. KefB and KefC are GSH-gated K+ efflux systems that are activated by the formation and binding of glutathione adducts that are generated during detoxification.

Derivatization of phosphopeptides with mercapto- and amino-functionalized conjugate groups by phosphate elimination and subsequent Michael addition

Kinetics of the beta-elimination of the phosphate group from H-Tyr-Ser(PO3H2)-Phe-OH and H-Tyr-Thr(PO3H2)-Phe-OH and subsequent addition of thiols and amines to the dehydroalaninyl and beta-methyldehydroalaninyl residues formed, were followed by RP HPLC under alkaline conditions in the absence and presence of Ba2+ ions. By this reaction sequence, the phosphoserinyl peptide was conjugated with mono-N-(2-mercaptoethyl)amide of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (4), a mercapto-functionalized pentapeptide, H-His-Gly-Gly-His-Gly-NH(CH2)4SH, and an amino-functionalized fluorescent dye, 5-dimethylaminonaphthalene-1-[N-(5-aminopentyl)]sulfonamide (dansyl cadaverine). The beta-methyldehydroalanine residue was, in turn, observed to be a poor Michael acceptor.

Lanthanide-mediated phosphoester hydrolysis and phosphate elimination from phosphopeptides

Lanthanide ions can mediate both phosphomonoester hydrolysis and beta-elimination of inorganic phosphate from polypeptide substrates under near-physiological conditions of pH, temperature, and salt.

A Comparison of the Microstructural Feature and Bonding Strength of Plasma-Sprayed Hydroxyapatite Coatings with Hydrothermal and Vacuum Post-Heat Treatment

Plasma-sprayed hydroxyapatite (Ca10(PO4)6(OH)2, HA) is an attractive biomaterials because it can provide the osteoconductivity and osseointegration to form a chemical bonding to bone. In order to improve the properties of HA, the post-heat treatment was performed and the effect of an ambient atmosphere on phase purity, crystallinity and bonding strength of plasma-sprayed HA coatings (HACs) was investigated. On the basis of quantitative analysis in crystallinity and phase content, autoclaving hydrothermal treatment was found to be effective for the elimination of impurity phases and amorphous calcium phosphate. It should be noted that plasma-sprayed HACs with a crystallinity of 84.5% and very low impurity phase content of 1.4 mass% can be obtained through a low temperature (200°C) hydrothermal treatment. Experimental evidence confirmed that the saturated steam atmosphere plays an important role in lowering the heating temperatures and promoting HA crystallization. Furthermore, hydrothermal-treated HACs show superiority in prominent OH− and PO43− groups than vacuum heating HACs. In addition, the bonding strength of the coating layer also can be improved from 35.7 MPa (vacuum heating) to 39.4 MPa (150°C hydrothermal treatment). From the evaluation of microstructural features and properties, an excessively high heating temperature will cause contraction with HA crystallization resulting in detrimental large cracks that lead to bonding degradation. Consequently, hydrothermal treatment seems very promising as a way of improving the properties of HA coatings.

Stereospecificity of hydride transfer in NAD +-catalyzed 2-deoxy- scyllo-inosose synthase, the key enzyme in the biosynthesis of 2-deoxystreptamine-containing aminocyclitol antibiotics

The key enzyme in the biosynthesis of clinically important aminocyclitol antibiotics is 2-deoxy- scyllo-inosose synthase (DOIS), which converts ubiquitous d-glucose 6-phosphate (G-6-P) into the specific carbocycle, 2-deoxy- scyllo-inosose with an aid of NAD +–NADH recycling. The NAD +-dependent first step of the DOIS reaction was examined in detail by the use of 6-phosphonate and 6-homophosphonate analogs of G-6-P. Both analogs showed competitive inhibition against the DOIS reaction with K i values of 1.3 and 2.8 mM, respectively, due to their inability for the subsequent phosphate elimination. Based on the direct spectrophotometric observation of NADH formed by the hydride transfer from 6-phosphonate to NAD +, the stereospecificity of the hydride transfer in the DOIS reaction was analyzed with 6-[4- 2H]phosphonate and was found to be pro- R specific.

Derivatization of phosphorylated peptides with S- and N-nucleophiles for enhanced ionization efficiency in matrix-assisted laser desorption/ionization mass spectrometry.

The identification of phosphorylation sites is essential for a full understanding of the cellular functions of proteins. However, mass spectrometric analysis is often hampered by the low abundance of phosphoproteins, the difficulty of obtaining full sequence coverage by specific proteolysis reactions, and the low ionization efficiency of phosphopeptides compared with their non-phosphorylated analogs. In the present work a beta-elimination/Michael addition was used to replace the phosphate groups of pSer or pThr by a group which gives rise to an enhanced ionization efficiency. In order to find optimum reaction conditions, beta-elimination/Michael addition was examined using phosphorylated model peptides. Whereas complete elimination of phosphate could be achieved by treatment with barium hydroxide in organic solvents such as ethanol or acetonitrile, the yield of the Michael adduct strongly depended on the nucleophile and the peptide sequence. Reaction with 2-phenylethanethiol, p-bromophenethylamine and ethylenediamine clearly resulted in products showing higher matrix-assisted laser desorption/ionization (MALDI) signal intensities compared with those of the corresponding phosphorylated precursors. The method was successfully used to identify phosphorylated sequences of ovalbumin and human Stat1 by in-gel derivatization with 2-phenylethanethiol and subsequent peptide mass fingerprint analysis of the trypsin digests.

Production of cellulase by Trichoderma reesei from dairy manure

Cellulase production by the fungi Trichoderma reesei was studied using dairy manure as a substrate. Data showed that T. reesei RUT-C30 had higher cellulase production than T. reesei QM 9414 and that a homogenized manure, treated by a blender to reduce fiber size, led to higher cellulase production. The cellulase production was further optimized by growing T. reesei RUT-C30 on homogenized manure. The effects of manure concentration, pH, and temperature on cellulase production were investigated with optimal parameter values determined to be 10 g/l manure (dry basis), 25.5 °C, and pH 5.7, respectively. Elimination of CaCl 2, MgSO 4, nitrogen sources (NH 4 + and urea) and trace elements (Fe 2+, Zn 2+, Co 2+ and Mn 2+) from the original salt solution had no negative influence on the cellulase production, while phosphate elimination did reduce cellulase production. Based on above results, the final medium composition was simplified with manure additives being KH 2PO 4, tween-80 and CoCl 2 only. Using this medium composition and a reaction time of 6–8 days, a maximum cellulase production activity of 1.74 IU/ml of filter paper activity, 12.22 IU/ml of CMCase activity, and 0.0978 IU/ml of β-glucosidase was obtained. This filter paper activity is the highest ever reported in cellulase production from agricultural wastes.

Effects of Biphenol and Quinone Derivatives on the Radiation-Initiated Dephosphorylation of 1-Glycerophosphate

The effects of biphenol and quinone derivatives on the radiation-initiated dephosphorylation of 1-glycerophosphate under various conditions were studied. It was found that in deaerated media, para-and ortho-quinone derivatives inhibited the formation of the phosphate ion by a factor of 1.8–1.9, and hydroquinone and pyrocatechol derivatives inhibited this reaction by a factor of 1.3–1.4, whereas 5-methylresorcinol and 2-methoxyphenol had no effect on the elimination of inorganic phosphate. It was demonstrated that a decrease in the radiation-chemical yields of the phosphate anion was due to the oxidation of 1-glycerophosphate radicals, which were formed under γ-radiolysis in aqueous solutions, by quinones.