A continuous kinetic assay for adenylation enzyme activity and inhibition

Adenylation/adenylate-forming enzymes catalyze the activation of a carboxylic acid at the expense of ATP to form an acyl-adenylate intermediate and pyrophosphate (PP_i). In a second half-reaction, adenylation enzymes catalyze the transfer of the acyl moiety of the acyl-adenylate onto an acceptor molecule, which can be either a protein or a small molecule. We describe the design, development, and validation of a coupled continuous spectrophotometric assay for adenylation enzymes that employs hydroxylamine as a surrogate acceptor molecule, leading to the formation of a hydroxamate. The released pyrophosphate from the first half-reaction is measured using the pyrophosphatase-purine nucleoside phosphorylase coupling system with the chromogenic substrate 7-methylthioguanosine (MesG). The coupled hydroxamate-MesG assay is especially useful for characterizing the activity and inhibition of adenylation enzymes that acylate a protein substrate and/or fail to undergo rapid ATP-PP_i exchange.     

Discovery of PPi-type Phosphoenolpyruvate Carboxykinase Genes in Eukaryotes and Bacteria

Phosphoenolpyruvate carboxykinase (PEPCK) is one of the pivotal enzymes that regulates the carbon flow of the central metabolism by fixing CO₂ to phosphoenolpyruvate (PEP) to produce oxaloacetate or vice versa. Whereas ATP- and GTP-type PEPCKs have been well studied, and their protein identities are established, inorganic pyrophosphate (PP_i)-type PEPCK (PP_i-PEPCK) is poorly characterized. Despite extensive enzymological studies, its protein identity and encoding gene remain unknown. In this study, PP_i-PEPCK has been identified for the first time from a eukaryotic human parasite, Entamoeba histolytica, by conventional purification and mass spectrometric identification of the native enzyme, followed by demonstration of its enzymatic activity. A homolog of the amebic PP_i-PEPCK from an anaerobic bacterium Propionibacterium freudenreichii subsp. shermanii also exhibited PP_i-PEPCK activity. The primary structure of PP_i-PEPCK has no similarity to the functional homologs ATP/GTP-PEPCKs and PEP carboxylase, strongly suggesting that PP_i-PEPCK arose independently from the other functional homologues and very likely has unique catalytic sites. PP_i-PEPCK homologs were found in a variety of bacteria and some eukaryotes but not in archaea. The molecular identification of this long forgotten enzyme shows us the diversity and functional redundancy of enzymes involved in the central metabolism and can help us to understand the central metabolism more deeply.     

A new, convenient method for the rapid analysis of inorganic pyrophosphate.

A new method for the rapid analysis of inorganic pyrophosphate (PP_i) which utilizes the enzyme ATP sulfurylase is described. All components of the assay system are commercially available and inexpensive. The assay is linear over the range of 0.5–50.0 nmol of PP_i and is not affected by inorganic phosphate. ATP and PP_i can both be analyzed using this method.     

Nonribosomal peptide synthetases-evidence for a second ATP-binding site

δ-(l-α-Aminoadipyl)-l-cysteinyl-d-valine synthetase (ACVS) catalyses, via the protein thiotemplate mechanism, the nonribosomal biosynthesis of the penicillin and cephalosporin precursor tripeptide δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (ACV). The complete and fully saturated biosynthetic system approaches maximum rate of product generation with increasing ATP concentration. Nonproductive adenylation of ACVS, monitored utilising the ATP–[³²P]PP_i exchange reaction, has revealed substrate inhibition with ATP. The kinetic inhibition pattern provides evidence for the existence of a second nucleotide-binding site with possible implication in the regulatory mechanism. Under suboptimal reaction conditions, in the presence of MgATP^2?, l-Cys and inorganic pyrophosphatase, ACVS forms adenosine(5′)tetraphospho(5′)adenosine (Ap₄A) from the reverse reaction of adenylate formation involving a second ATP molecule. The potential location of the second ATP binding site was deduced from sequence comparisons and molecular visualisation in conjunction to data obtained from biochemical analysis.     

PPiase-Activated ATP-Dependent H+ Transport at the Tonoplast of Mesophyll Cells of the CAM Plant Kalanchoë daigremontiana*

The co-ordinated action of the two proton-transporting enzymes at the tonoplast of the CAM plants. daigremontiana, viz. the ATPase and the PP_iase, was studied by measuring fluorescent dye quenching. The initial rates of ATP and PP_i-dependent H⁺ transport into tonoplast vesicles were additive, i.e. the sum of the rates obtained with each substrate alone was in the range obtained with both substrates added together at the same time. Conversely, the activities of the two H⁺ pumps were non-additive in establishing the steady-state level, indicating that the final steady state was under thermodynamic control of a maximal attainable proton gradient. The initial rates of ATP-dependent H⁺ transport were stimulated enormously if ATP was added a few minutes after pre-energization of the vesicles with PP_i. This stimulation was observed only when the PP_iase was active. A similar effect was not found for PP_i-dependent H⁺ transport after pre-energization with ATP. Hence, a PP_iase-activated ATP-dependent H⁺ transport can be distinguished from the basic ATP- and the basic PP_i-dependent H⁺ transport. In parallel a PP_i-dependent stimulation of ATP hydrolysis in the absence of ionophores was measured, which can only be attributed to the activity of the PP_iase. PP_iase-activated ATP-dependent H⁺ transport depends on the presence of permeant anions. It shows properties of both H⁺ transport activities, i.e. the chloride and malate stimulation and the DCCD inhibition of the ATP-dependent H⁺ transport activity, the nitrate stimulation and the KF inhibition of the PP_i-dependent H⁺ transport activity. Only MgPP_i and MgATP were effective as the respective substrates. The PP_iase-activated ATP-dependent H⁺ transport had a half life of about 5–9 minutes. It is concluded that the PP_iase may play an important role in kinetic regulation of the ATPase, and implications for CAM metabolism are discussed.     

A Cationic Cluster of Amino Acid Residues of Inorganic Pyrophosphatase from <Emphasis Type="Italic">Escherichia coli</Emphasis> as a Possible Site of Effector Binding

A computer-assisted analysis of the molecule of Escherichia coli pyrophosphatase was earlier used to localize the site capable of binding free pyrophosphate (PP_i) or methylenediphosphonate, a PP_i analogue, and thereby activating the enzyme. A cluster of positively charged amino acid residues (Lys146, Lys148, Lys115, and Arg43) was revealed, and Lys115Ala, Lys148Gln, and Arg43Gln mutant pyrophosphatases (PPases) were obtained. It was shown that the kinetics of hydrolysis of the magnesium pyrophosphate (MgPP_i) substrate by these mutant variants does not obey the Michaelis-Menten equation, which is expressed in two slopes in the double-reciprocal plots of the enzyme reaction rate vs. substrate concentration. The two regions on the curves correspond to the ranges of high and low MgPP_i concentrations. This suggests that, in all mutant variants of the enzyme, the binding of PP_i at the effector site weakens, whereas the affinity of MgPP_i for the active site remains practically unchanged. Other properties of the enzymes, such as their oligomeric states, resistance to thermal denaturation, and resistance to the denaturing agent guanidine hydrochloride, were thoroughly studied. The constants of binding of Mg²⁺ to mutant enzymes in the absence of substrate and to enzyme-substrate complexes were determined. The introduction of amino acid substitutions was shown to stabilize the protein globule.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 251–258.Original Russian Text Copyright © 2005 by Sitnik, Avaeva.     

Rapid and selective enzymatic assay for l-methionine based on a pyrophosphate detection system

An enzymatic assay for l-methionine was developed by coupling adenosylmethionine synthetase (AdoMetS) to a pyrophosphate (PP_i) detection system, which was constructed using pyruvate, phosphate dikinase. To expand the use of this assay, the PP_i detection system was embodied as three different forms, which allowed PP_i to be measured by UV, visible, and fluorescent light detectors. The assay system was robust and could tolerate the addition of inorganic phosphate and ATP to the assay mixtures. l-Methionine could be accurately determined by coupling the PP_i detection system and AdoMetS. This AdoMetS coupling assay was highly selective to l-methionine and exhibited no significant activity to other proteinaceous amino acids, ammonia, or urea, unlike conventional enzymatic assays for l-methionine. Spike and recovery tests showed that the AdoMetS assay could accurately and reproducibly determine increases in l-methionine in human plasma samples without any pretreatment to remove proteins and potentially interfering low-molecular-weight molecules. The high selectivity and robustness of the AdoMetS assay provide rapid and high-throughput analysis of l-methionine in various kinds of analytes.     

32P-labeling of bovine tryptophanyl-tRNA synthetase with [32P]pyrophosphate

The covalent derivative of the tryptophanyl-tRNA synthetase obtained under the action of³²PP_i contains one mole of the covalently bound pyrophosphate (or 2 moles of orthophosphate) per mole of dimeric enzyme. Dephosphorylation with alkaline phosphatase causes practically no changes of enzymatic activity although the enzyme looses its ability to bind PP_i.Enzymes tryptophanyl-tRNA synthetase (EC 6.1.1.2), alkaline phosphatase (EC 3.1.3.1), inorganic pyrophosphatase (EC 3.6.1.1)     

Catalysis by isolated β-subunits of the ATP Synthase/ATPase from Thermophilic bacillus PS3. Hydrolysis of Pyrophosphate

Although the capacity of isolated β-subunits of the ATP synthase/ATPase to perform catalysis has been extensively studied, the results have not conclusively shown that the subunits are catalytically active. Since soluble F₁ of mitochondrial H⁺-ATPase can bind inorganic pyrophosphate (PP_i) and synthesize PP_i from medium phosphate, we examined if purified His-tagged β-subunits from Thermophilic bacillus PS3 can hydrolyze PP_i. The difference spectra in the near UV CD of β-subunits with and without PP_i show that PP_i binds to the subunits. Other studies show that β-subunits hydrolyze [³²P] PP_i through a Mg²⁺-dependent process with an optimal pH of 8.3. Free Mg²⁺ is required for maximal hydrolytic rates. The Km for PP_i is 75 μM and the Vmax is 800 pmol/min/mg. ATP is a weak inhibitor of the reaction, it diminishes the Vmax and increases the Km for PP_i. Thus, isolated β-subunits are catalytically competent with PP_i as substrate; apparently, the assembly of β-subunits into the ATPase complex changes substrate specificity, and leads to an increase in catalytic rates.     

Bimodular Peptide Synthetase SidE Produces Fumarylalanine in the Human Pathogen Aspergillus fumigatus

The filamentous mold Aspergillus fumigatus causes invasive aspergillosis, a potentially life-threatening infectious disease, in humans. The sidE gene encodes a bimodular peptide synthetase and was shown previously to be strongly upregulated during initiation of murine lung infection. In this study, we characterized the two adenylation domains of SidE with the ATP-[³²P]pyrophosphate exchange assay in vitro, which identified fumarate and l-alanine, respectively, as the preferred substrates. Using full-length holo-SidE, fumarylalanine (FA) formation was observed in vitro. Furthermore, FA was identified in A. fumigatus culture supernatants under inducing conditions, unless sidE was genetically inactivated. As FA is structurally related to established pharmaceutical products exerting immunomodulatory activity, this work may contribute to our understanding of the virulence of A. fumigatus.     

Enzymatic biosynthesis of ergotamine and investigation on some aminoacyl-tRNA synthetases in Claviceps

An enzyme system from Claviceps purpurea (Fr.) Tul. catalyzing the incorporation of l-phenylalanine into ergotamine - ergotamine synthetase - was purified 172-fold. This was done by a combination of ammonium sulfate precipitation, gel filtration, ion-exchange chromatography on DEAE-Sepharose CL-6B, and hydroxyapatite chromatography. The activation of ergotamine specific amino acids as well as d-lysergic acid and dihydrolysergic acid via adenylates, as determined by the ATP-³²PP_i exchange, was investigated. Phenylalanyl-tRNA synthetase, catalyzing the same type of activation reaction, could not be separated from ergotamine synthetase by the purification procedure applied. Therefore, at the present stage of enzyme purification, phenylalanine-dependent ATP-³²PP_i exchange cannot be used to measure ergotamine synthetase activity specifically.Phenylalanyl-tRNA synthetase and leucyl-tRNA synthetase were separated into mitochondrial and cytoplasmic isoenzymes by hydroxyapatite chromatography. Their charging activities of procaryotic versus eucaryotic tRNA and their molecular masses were determined.     

Inhibition of photosynthesis in isolated spinach chloroplasts by inorganic phosphate or inorganic pyrophosphatase in the presence of pyrophosphate and magnesium ions

Inhibition of photosynthesis in isolated spinach chloroplasts by P_i is decreased by the presence of PP_i and increased with increasing Mg²⁺ concentration. Previously reported regulation of this photosynthesis by protein factors from spinach leaves appears to be due mostly to pyrophosphate phosphohydrolase (EC 3.6.1.1) activity which converts PP_i to P_i and to the effects of PP_i and Mg²⁺ on this pyrophosphatase activity.     

A requirement for chelation in obtaining functional chloroplasts of sunflower and wheat.

In studying conditions for obtaining photosynthetically functional chloroplasts from mesophyll protoplasts of sunflower and wheat, a strong requirement for chelation was found. The concentration of chelator, either EDTA or pyrophosphate (PP_i), required for maximum activation depended on the pH, the concentration of orthophosphate (P_i) in the assay, and the chelator used. Studies with EDTA indicate that including the chelator in the isolation, resuspension, and assay media, in the absence of divalent cations, was most effective. Increased concentration of EDTA from 1 to 10 mm broadened the pH response curve for photosynthesis, inasmuch as a higher concentration of chelator was required for activation of photosynthesis at lower pH.Either EDTA, PP_i, or citrate could activate photosynthesis of sunflower chloroplasts isolated and assayed at pH 8.4. At pH 7.6, PP_i and EDTA were equally effective at low P_i concentrations but PP_i was particularly effective in shortening the induction period at high concentrations of P_i (2.5 mm) in the assay medium. Including 1 mm 3-phosphoglycerate in the assay medium with or without P_i could not replace the need for chelation. However, 3-phosphoglycerate + EDTA in the assay medium with 0.5 mm P_i, pH 7.6, gave a short induction period and rates of photosynthesis similar to those with 10 mm PP_i. The results suggest that PP_i can have a dual effect at the lower pH through chelation and inhibition of the phosphate transporter.Photosynthesis by sunflower chloroplasts isolated and assayed at pH 8.4 with 0.2 mm EDTA (+ 0.5 mm P_i in the assays) was severely inhibited by 2 mM CaCl₂, MgCl₂, or MnCl₂. Wheat chloroplasts isolated and assayed at pH 8.4 without chelation, and assayed with 0.2 mm P_i, had low rates of photosynthesis (25 μmol O₂ evolved mg^?1 chlorophyll h^?1) which were strongly inhibited by 2 to 4 mm MgCl₂, MnCl₂, or CaCl₂. With inclusion of EDTA and P_i at optimum levels, isolated chloroplasts of sunflower and wheat have high rates of photosynthesis and PP_i or divalent cations are not of benefit.     

A method for the concentration and for the colorimetric determination of nanomoles of inorganic pyrophosphate

A generally applicable, inexpensive, and sensitive method for the determination of inorganic pyrophosphate (PP_i) was developed. PP_i was quantitatively separable from solution even in nanomolar concentrations by filtration through a membrane filter in the presence of CaCl₂ and KF. The separated PP_i was dissolved by immersing the filter in 0.5 n H₂SO₄. Inorganic phosphate (P_i) was removed by precipitating it as a phosphomolybdate-triethylamine complex and the PP_i was measured as a green pyrophosphomolybdate in the presence of 2-mercaptoethanol. Nucleotides and phosphate esters do not react. PP_i can be accurately assayed even when there is a 10⁴-fold excess of P_i. Trimetaphosphate, tripolyphosphate, and tetrapolyphosphate also give this green color, but the rate of the color formation is 50 times slower than that with PP_i. Thus this interference of the polyphosphates can be eliminated or the polyphosphates can be assayed simultaneously with the PP_i in the same sample.     

The Landscape of Recombination Events That Create Nonribosomal Peptide Diversity

Nonribosomal peptides (NRP) are crucial molecular mediators in microbial ecology and provide indispensable drugs. Nevertheless, the evolution of the flexible biosynthetic machineries that correlates with the stunning structural diversity of NRPs is poorly understood. Here, we show that recombination is a key driver in the evolution of bacterial NRP synthetase (NRPS) genes across distant bacterial phyla, which has guided structural diversification in a plethora of NRP families by extensive mixing and matching of biosynthesis genes. The systematic dissection of a large number of individual recombination events did not only unveil a striking plurality in the nature and origin of the exchange units but allowed the deduction of overarching principles that enable the efficient exchange of adenylation (A) domain substrates while keeping the functionality of the dynamic multienzyme complexes. In the majority of cases, recombination events have targeted variable portions of the A_core domains, yet domain interfaces and the flexible A_sub domain remained untapped. Our results strongly contradict the widespread assumption that adenylation and condensation (C) domains coevolve and significantly challenge the attributed role of C domains as stringent selectivity filter during NRP synthesis. Moreover, they teach valuable lessons on the choice of natural exchange units in the evolution of NRPS diversity, which may guide future engineering approaches.     

Physiologic and pathologic functions of the NPP nucleotide pyrophosphatase/phosphodiesterase family focusing on NPP1 in calcification

The catabolism of ATP and other nucleotides participates partly in the important function of nucleotide salvage by activated cells and also in removal or de novo generation of compounds including ATP, ADP, and adenosine that stimulate purinergic signaling. Seven nucleotide pyrophosphatase/phosphodiesterase NPP family members have been identified to date. These isoenzymes, related by up conservation of catalytic domains and certain other modular domains, exert generally non-redundant functions via distinctions in substrates and/or cellular localization. But they share the capacity to hydrolyze phosphodiester or pyrophosphate bonds, though generally acting on distinct substrates that include nucleoside triphosphates, lysophospholipids and choline phosphate esters. PP_i generation from nucleoside triphosphates, catalyzed by NPP1 in tissues including cartilage, bone, and artery media smooth muscle cells, supports normal tissue extracellular PP_i levels. Balance in PP_i generation relative to PP_i degradation by pyrophosphatases holds extracellular PP_i levels in check. Moreover, physiologic levels of extracellular PP_i suppress hydroxyapatite crystal growth, but concurrently providing a reservoir for generation of pro-mineralizing P_i. Extracellular PP_i levels must be supported by cells in mineralization-competent tissues to prevent pathologic calcification. This support mechanism becomes dysregulated in aging cartilage, where extracellular PP_i excess, mediated in part by upregulated NPP1 expression stimulates calcification. PP_i generated by NPP1modulates not only hydroxyapatite crystal growth but also chondrogenesis and expression of the mineralization regulator osteopontin. This review pays particular attention to the role of NPP1-catalyzed PP_i generation in the pathogenesis of certain disorders associated with pathologic calcification.     

Identification of new protein complexes of Escherichia coli inorganic pyrophosphatase using pull-down assay

Inorganic pyrophosphatase (PPase) is a conserved and essential enzyme catalyzing the hydrolysis of pyrophosphate PP_i. Its activity is required to promote a lot of thermodynamically unfavorable reactions including biosynthesis of activated precursors of sugars and amino acids. Several protein partners of PPase were found so far in Escherichia coli by large-scale approaches. Functional role of these interactions was not studied. In this paper we report the identification of three protein partners of E. coli PPase not found earlier. Pull-down assay on the Ni²⁺-chelating column using 6His-tagged PPase as bait was used to isolate PPase complexes from stationary-phase cells. Of several isolated protein components, five were identified by MALDI-TOF mass-spectrometry: two chaperones (DnaK and GroEL) and three enzymes of carbohydrate and amino acid metabolism (FbaB, fructose-1,6-bisphosphate aldolase, class I; GadA, l-glutamate decarboxylase; and KduI, 5-keto-4-deoxyuronate isomerase). These three proteins were cloned, expressed and purified in 6His-tagged and/or tag-free forms. Their binary interactions with PPase were verified by independent approaches. Initial characterization of the complexes indicates that PPase may stabilize its protein partners against unfolding or degradation. Comparative analysis of the PPase protein partners allowed an insight into its possible involvement in the cell metabolic regulation.     

In vitro construction of the tufB-lacZ fusion: analysis of the regulatory mechanism of tufB promoter

Summary Genetic studies suggest that the so-called phosphorus-family of enzymes inN. crassa are controlled by a complex system of regulatory genes which are responsive to the level of phosphorus in the growth medium. The intracellular metabolite(s) that interact with this system to signal changes in the external phosphorus concentration has not been identified. In this study the pools of acid-soluble, phosphorus-containing, compounds are measured in wild-type and phosphorus-family enzyme regulatory mutant strains ofN. crassa before and during phosphorus starvation.Prolonged phosphorus starvation of wild-typeN. crassa failed to alter significantly the pre-starvation level of intracellular orthophosphate, suggesting that intracellular P_i would be a poor effector signal for the control of the phosphorus family enzymes. However, inorganic pyrophosphate (PP_i) decreased 15-fold, and tri- and tetrapolyphosphate (PPP_i and PPPP_i) increased 3- to 5-fold within 15 minutes after transfer of the wild-type strain to phosphorus-free medium. Phosphate starvation of seven different regulatory gene mutant strains resulted in a rapid decrease in the PP_i pool similar to that which occurred in the wild-type. However, only two of these seven strains showed increased PPP_i and PPPP_i pools following phosphate starvation. Additional experiments demonstrated that PP_i pools, but not PPP_i and PPPP_i pools, were unaffected by several starvation regimens other than phosphorus starvation. Metabolic studies employing H₃ ³²PO₄ showed that the pool of PP_i was labeled to steady-state levels after two minutes of continuous labeling of a phosphate-sufficient culture. Furthermore, long-term steady-state labeling showed that the intracellular PP_i pool was directly responsive to the decrease in the extracellular P_i concentration of the medium resulting from cell growth. Growth on phosphoethanolamine, a phosphorus source that allows a modest degree of derepression even in growing cells, resulted in lower levels of PP_i than were seen in phosphate-grown cells. These observations suggest that PP_i may be involved in the mechanism responsible for the control of phosphorus-family enzyme regulatory gene product activity.