Role of the interaction between troponin T and AMP deaminase by zinc bridge in modulating muscle contraction and ammonia production.

The N-terminal region of troponin T (TnT) does not bind any protein of the contractile machinery and the role of its hypervariability remains uncertain. In this review we report the evidence of the interaction between TnT and AMP deaminase (AMPD), a regulated zinc enzyme localized on the myofibril. In periods of intense muscular activity, a decrease in the ATP/ADP ratio, together with a decrease in the tissue pH, is the stimulus for the activation of the enzyme that deaminating AMP to IMP and NH3 displaces the myokinase reaction towards the formation of ATP. In skeletal muscle subjected to strong tetanic contractions, a calpain-like proteolytic activity produces the removal in vivo of a 97-residue N-terminal fragment from the enzyme that becomes desensitized towards the inhibition by ATP, leading to an unrestrained production of NH3. When a 95-residue N-terminal fragment is removed from AMPD by trypsin, simulating in vitro the calpain action, rabbit fast TnT or its phosphorylated 50-residue N-terminal peptide binds AMPD restoring the inhibition by ATP. Taking in consideration that the N-terminus of TnT expressed in human as well as rabbit white muscle contains a zinc-binding motif, we suggest that TnT might mimic the regulatory action of the inhibitory N-terminal domain of AMPD due to the presence of a zinc ion connecting the N-terminal and C-terminal regions of the enzyme, indicating that the two proteins might physiologically associate to modulate muscle contraction and ammonia production in fast-twitching muscle under strenuous conditions.

Structure of Arabidopsis thaliana N6-methyl-AMP deaminase ADAL with bound GMP and IMP and implications for N6-methyl-AMP recognition and processing.

Arabidopsis thaliana aminohydrolase (AtADAL) has been shown to be involved in the metabolism of N6-methyl-AMP, a proposed intermediate during m6A-modified RNA metabolism, which can be subsequently incorporated into newly synthesized RNA by Pol II. It has been proposed that AtADAL will prevent N6-methyl-AMP reuse and catabolize it to inosine monophosphate (IMP). Here, we have solved the crystal structures of AtADAL in the apo form and in complex with GMP and IMP in the presence of Zn2+. We have identified the substrate-binding pocket of AtADAL and compared it with that for adenosine deaminase (ADA), adenine deaminase (ADE) and AMP deaminase (AMPD) from multiple species. The comparisons reveal that plant ADAL1 may have the potential ability to catalyze different alkyl-group substituted substrates.

Biochemical and physiological investigations on adenosine 5' monophosphate deaminase from Plasmodium spp.

The interplay between ATP generating and utilizing pathways in a cell is responsible for maintaining cellular ATP/energy homeostasis that is reflected by Adenylate Energy Charge (AEC) ratio. Adenylate kinase (AK), that catalyzes inter-conversion of ADP, ATP and AMP, plays a major role in maintaining AEC and is regulated by cellular AMP levels. Hence, the enzymes AMP deaminase (AMPD) and nucleotidases, which catabolize AMP, indirectly regulate AK activity and in-turn affect AEC. Here, we present the first report on AMPD from Plasmodium, the causative agent of malaria. The recombinant enzyme expressed in Saccharomyces cerevisiae was studied using functional complementation assay and residues vital for enzyme activity have been identified. Similarities and differences between Plasmodium falciparum AMPD (PfAMPD) and its homologs from yeast, Arabidopsis and humans are also discussed. The AMPD gene was deleted in the murine malaria parasite P. berghei and was found to be dispensable during all stages of the parasite life cycle. However, when episomal expression was attempted, viable parasites were not obtained, suggesting that perturbing AMP homeostasis by over-expressing AMPD might be lethal. As AMPD is known to be allosterically modulated by ATP, GTP and phosphate, allosteric activators of PfAMPD could be developed as anti-parasitic agents.

Alternative conformation induced by substrate binding for Arabidopsis thalianaN6-methyl-AMP deaminase.

Adenosine deaminase is involved in adenosine degradation and salvage pathway, and plays important physiological roles in purine metabolism. Recently, a novel type of adenosine deaminase-like protein has been identified, which displays deamination activity toward N6-methyl-adenosine monophosphate but not adenosine or AMP, and was consequently named N6-methyl-AMP deaminase (MAPDA). The underlying structural basis of MAPDA recognition and catalysis is poorly understood. Here, we present the crystal structures of MAPDA from Arabidopsis thaliana in the free and in the ligand-bound forms. The protein contains a conserved (ß/α)8 Tim-barrel domain and a typical zinc-binding site, but it also exhibits idiosyncratic local differences for two flexible helices important for substrate binding. The extensive interactions between the N6-methyl-AMP substrate or the inosine monophosphate product and the enzyme were identified, and subsequently evaluated by the deamination activity assays. Importantly, each structure reported here represents a different stage of the catalytic pathway and their structural differences suggested that the enzyme can exist in two distinct conformational states. The open state switches to the closed one upon the binding of ligands, brought about by the two critical helices. Our structural studies provide the first look of this important metabolic enzyme and shed lights on its catalytic pathway, which holds promise for the structure-based drug design for MAPDA-related diseases.

Role of the HPRG Component of Striated Muscle AMP Deaminase in the Stability and Cellular Behaviour of the Enzyme.

Multiple muscle-specific isoforms of the Zn2+ metalloenzyme AMP deaminase (AMPD) have been identified based on their biochemical and genetic differences. Our previous observations suggested that the metal binding protein histidine-proline-rich glycoprotein (HPRG) participates in the assembly and maintenance of skeletal muscle AMP deaminase (AMPD1) by acting as a zinc chaperone. The evidence of a role of millimolar-strength phosphate in stabilizing the AMPD-HPRG complex of both AMPD1 and cardiac AMP deaminase (AMPD3) is suggestive of a physiological mutual dependence between the two subunit components with regard to the stability of the two isoforms of striated muscle AMPD. The observed influence of the HPRG content on the catalytic behavior of the two enzymes further strengthens this hypothesis. Based on the preferential localization of HPRG at the sarcomeric I-band and on the presence of a Zn2+ binding motif in the N-terminal regions of fast TnT and of the AMPD1 catalytic subunit, we advance the hypothesis that the Zn binding properties of HPRG could promote the association of AMPD1 to the thin filament.

Ability of HMGB1 protein to bind to intrinsically bent and non-bent DNA sites in the AMPD2 gene amplicon.

HMGB-like proteins are architectural chromatin factors, and their function is heavily dependent on their ability to interact with DNA (especially non-canonical DNA structures). HMGB1 is involved in many DNA processes, and dysregulation of HMGB protein expression has profound effects on cellular transcription, resulting in severe developmental defects as well as cancer. During DNA replication, elements that form the origin are still not well defined in metazoans. Sites with A (adenine) or T (thymine) repeats cause intrinsic curvatures in the DNA and are described to be involved in the replication machinery by providing binding sites to replication proteins. As a result, the DNA molecule shows intrinsically bent DNA sites, caused by periodic repeats of 2 or more As/Ts (dA/dT) as well as intrinsically non-bent DNA sites (INBDs), due to a succession of curvatures that cancel each other. In the present study, we mapped 11 INBDSs present in the AMPD2 gene that are related to each replication origin (oriGNAI3, oriC, oriB, and oriA). Following characterization of INBDSs, we tested the ability of HMGB1 to bind to the bent (b1, b2, b4a, b4b, b5, b6, b7, and b8) and non-bent DNA fragments (nb7, nb11, nb1, nb2, nb4, and nb5) via electrophoretic mobility shift assays. All fragments showed efficient binding to HMGB1. However, the non-bent DNA fragments nb2, nb4, and nb5 showed slightly reduced binding efficiency.

AMPD2 regulates GTP synthesis and is mutated in a potentially treatable neurodegenerative brainstem disorder.

Purine biosynthesis and metabolism, conserved in all living organisms, is essential for cellular energy homeostasis and nucleic acid synthesis. The de novo synthesis of purine precursors is under tight negative feedback regulation mediated by adenosine and guanine nucleotides. We describe a distinct early-onset neurodegenerative condition resulting from mutations in the adenosine monophosphate deaminase 2 gene (AMPD2). Patients have characteristic brain imaging features of pontocerebellar hypoplasia (PCH) due to loss of brainstem and cerebellar parenchyma. We found that AMPD2 plays an evolutionary conserved role in the maintenance of cellular guanine nucleotide pools by regulating the feedback inhibition of adenosine derivatives on de novo purine synthesis. AMPD2 deficiency results in defective GTP-dependent initiation of protein translation, which can be rescued by administration of purine precursors. These data suggest AMPD2-related PCH as a potentially treatable early-onset neurodegenerative disease.

Regulation of 5'-adenosine monophosphate deaminase in the freeze tolerant wood frog, Rana sylvatica.

BACKGROUND: The wood frog, Rana sylvatica, is one of a few vertebrate species that have developed natural freeze tolerance, surviving days or weeks with 65-70% of its total body water frozen in extracellular ice masses. Frozen frogs exhibit no vital signs and their organs must endure multiple stresses, particularly long term anoxia and ischemia. Maintenance of cellular energy supply is critical to viability in the frozen state and in skeletal muscle, AMP deaminase (AMPD) plays a key role in stabilizing cellular energetics. The present study investigated AMPD control in wood frog muscle. RESULTS: Wood frog AMPD was subject to multiple regulatory controls: binding to subcellular structures, protein phosphorylation, and effects of allosteric effectors, cryoprotectants and temperature. The percentage of bound AMPD activity increased from 20 to 35% with the transition to the frozen state. Bound AMPD showed altered kinetic parameters compared with the free enzyme (S0.5 AMP was reduced, Hill coefficient fell to approximately 1.0) and the transition to the frozen state led to a 3-fold increase in S0.5 AMP of the bound enzyme. AMPD was a target of protein phosphorylation. Bound AMPD from control frogs proved to be a low phosphate form with a low S0.5 AMP and was phosphorylated in incubations that stimulated PKA, PKC, CaMK, or AMPK. Bound AMPD from frozen frogs was a high phosphate form with a high S0.5 AMP that was reduced under incubation conditions that stimulated protein phosphatases. Frog muscle AMPD was activated by Mg.ATP and Mg.ADP and inhibited by Mg.GTP, KCl, NaCl and NH4Cl. The enzyme product, IMP, uniquely inhibited only the bound (phosphorylated) enzyme from muscle of frozen frogs. Activators and inhibitors differentially affected the free versus bound enzyme. S0.5 AMP of bound AMPD was also differentially affected by high versus low assay temperature (25 vs 5 degrees C) and by the presence/absence of the natural cryoprotectant (250 mM glucose) that accumulates during freezing. CONCLUSION: Maintenance of long term viability under the ischemic conditions in frozen muscle requires attention to the control of cellular energetics. Differential regulatory controls on AMPD by mechanisms including binding to muscle proteins, actions allosteric effectors, glucose and temperature effects and reversible phosphorylation adjust enzyme function for an optimal role in controlling cellular adenylate levels in ischemic frozen muscle. Stable modification of AMPD properties via freeze-responsive phosphorylation may contribute both to AMPD control and to coordinating AMPD function with other enzymes of energy metabolism in cold ischemic muscle.

Activity of adenosine deaminase and adenylate deaminase on adenosine and 2', 3(')-isopropylidene adenosine: role of the protecting group at different pH values.

The deamination rate of 2',3'-isopropylidene adenosine catalyzed by adenosine deaminase (ADA) from calf intestine and adenylate deaminase (AMPDA) from Aspergillus species has been evaluated and compared with that of the enzymatic reactions of adenosine, to elucidate the influence of the protecting group on enzyme activity.

2',3'-isopropylidene group, a molecular scaffold to study the activity of adenosine and adenylate deaminase on adenosine analogues modified in the ribose moiety.

2 ',3 '-Isopropylidene group can be used as a molecular scaffold for the introduction of modifications at 5 ' and 1 ' positions of adenosine and these modified nucleosides are used to evaluate the biocatalytic activity of adenosine and adenylate deaminase.

Characterization of the metallocenter of rabbit skeletal muscle AMP deaminase. A new model for substrate interactions at a dinuclear cocatalytic Zn site.

We have previously provided evidence for a dinuclear zinc site in rabbit skeletal muscle AMPD compatible with a (micro-aqua)(micro-carboxylato)dizinc(II) core with an average of two histidine residues at each metal site. XAS of the zinc binding site of the enzyme in the presence of PRN favors a model where PRN is added to the coordination sphere of one of the two zinc ions increasing its coordination number to five. The uncompetitive nature of the inhibition of AMPD by fluoride reveals that the anion probably displaces the nucleophile water molecule terminally coordinated to the catalytic Zn(1) ion at the enzyme C-terminus, following the binding of AMP at the Zn(2) ion located at N-terminus of the enzyme. Thus, the two Zn ions in the AMPD metallocenter operate together as a single catalytic unit, but have independent function, one of them (Zn(1)) acting to polarize the nucleophile water molecule, whilst the other (Zn(2)) acts transiently as a receptor for an activating substrate molecule. The addition of fluoride to AMPD also abolishes the cooperative behaviour induced in the enzyme by the inhibitory effect of ATP at acidic pH that probably resides in the competition with the substrate for an adenine nucleotide specific regulatory site located in the Zn(2) ion binding region and which is responsible for the positive homotropic cooperativity behaviour of AMPD.

Facile and rapid access to inosine puromycin analogues through the use of adenylate deaminase.

To study the ribosomal peptidyl transfer, puromycin analogues are of interest in which adenine has been replaced by hypoxanthine. We synthesized inosine puromycin analogues from 3'-azidodeoxyadenosine derivatives using adenylate deaminase for the quantitative transformation of the N-heterocycle. The amino acid coupling was carried out under Staudinger-Vilarrasa conditions in 94% yield starting from the protected and in 82% using the unprotected azide, thus, in the presence of two hydroxyls and a lactam function.

Characterization of the metallocenter of rabbit skeletal muscle AMP deaminase. Evidence for a dinuclear zinc site.

XAS of Zn-peptide binary and ternary complexes prepared using peptides mimicking the potential metal binding sites of rabbit skeletal muscle AMP deaminase (AMPD) strongly suggest that the region 48-61 of the enzyme contains a zinc binding site, whilst the region 360-372 of the enzyme is not able to form 1:1 complexes with zinc, in contrast with what has been suggested for the corresponding region of yeast AMPD. XAS performed on fresh preparations of rabbit skeletal muscle AMPD provides evidence for a dinuclear zinc site in the enzyme compatible with a (mu-aqua)(mu-carboxylato)dizinc(II) core with an average of two histidine residues at each metal site and a Zn-Zn distance of about 3.3 Angstrom. The data indicate that zinc is not required for HPRG/AMPD interaction, both zinc ions being bound to the catalytic subunit of the enzyme, one to the three conserved amino acid residues among those four assumed to be in contact with zinc in yeast AMPD, and the other at the N-terminal region, probably to His-52, Glu-53 and His-57. Tryptic digests of different enzyme preparations demonstrate the existence of two different protein conformations and of a zinc ion connecting the N-terminal and C-terminal regions of AMPD.

Deamination of 2',3'-O-isopropylideneadenosine-5'- carboxylic acid catalyzed by adenosine deaminase (ADA) and adenylate deaminase (AMPDA): influence of substrate ionization on the activity of the enzymes.

Adenosine deaminase (ADA) and adenylate deaminase (AMPDA) catalyze the deamination of 2 ',3 '-O-isopropylideneadenosine-5'-carboxylic acid to the corresponding inosine derivative and dependence of the rate of enzymatic reaction on the ionization degree of the substrate has been studied at different pH values.

Membrane association, mechanism of action, and structure of Arabidopsis embryonic factor 1 (FAC1).

Embryonic factor 1 (FAC1) is one of the earliest expressed plant genes and encodes an AMP deaminase (AMPD), which is also an identified herbicide target. This report identifies an N-terminal transmembrane domain in Arabidopsis FAC1, explores subcellular fractionation, and presents a 3.3-A globular catalytic domain x-ray crystal structure with a bound herbicide-based transition state inhibitor that provides the first glimpse of a complete AMPD active site. FAC1 contains an (alpha/beta)(8)-barrel characterized by loops in place of strands 5 and 6 that places it in a small subset of the amidohydrolase superfamily with imperfect folds. Unlike tetrameric animal orthologs, FAC1 is a dimer and each subunit contains an exposed Walker A motif that may be involved in the dramatic combined K(m) (25-80-fold lower) and V(max) (5-6-fold higher) activation by ATP. Normal mode analysis predicts a hinge motion that flattens basic surfaces on each monomer that flank the dimer interface, which suggests a reversible association between the FAC1 globular catalytic domain and intracellular membranes, with N-terminal transmembrane and disordered linker regions serving as the anchor and attachment to the globular catalytic domain, respectively.

[Family of AMP-deaminase genes]. / Rodzina genów deaminazy AMP.

AMP-deaminase (AMP-aminohydrolase, EC 3.5.4.6), a highly regulated oligomeric enzyme catalyzing the irreversible deamination of adenylic acid (5'-AMP), is located at a branch point of adenylate nucleotide catabolism. It plays an important role in the stabilization of adenylate energy charge (AEC) and the regulation of the purine nucleotide pool in several types of animal tissue. Tissue- and stage-specific isoforms of AMP-deaminase were described in mammals. In humans, three isozymes of AMP-deaminase, i.e. M (muscle), L (liver), and E (erythrocyte), exhibiting different physical, catalytic, and regulatory properties, were identified. AMP-deaminase activity is encoded by a multigene family in which two genes produce at least three mRNAs through alternative splicing of one of the primary transcripts. In this study we present all found and so far unpublished detailed knowledge about AMP-deaminase gene structures. We also present basic information on the effects of these gene mutations.

Calcium activates erythrocyte AMP deaminase [isoform E (AMPD3)] through a protein-protein interaction between calmodulin and the N-terminal domain of the AMPD3 polypeptide.

Erythrocyte AMP deaminase [isoform E (AMPD3)] is activated in response to increased intracellular calcium levels in Tarui's disease, following exposure of ionophore-treated cells to extracellular calcium, and by the addition of calcium to freshly prepared hemolysates. However, the assumption that Ca(2+) is a positive effector of isoform E is inconsistent with the loss of sensitivity to this divalent cation following dilution of erythrocyte lysates or enzyme purification. Ca(2+) regulation of isoform E was studied by examining in vitro effects of calmodulin (CaM) on this enzyme and by monitoring the influence of CaM antagonists on purine catabolic flow in freshly prepared erythrocytes under various conditions of energy imbalance. Erythrocyte and recombinant isoform E both adsorb to immobilized Ca(2+)-CaM, and relative adsorption across a series of N-truncated recombinant enzymes localizes CaM binding determinants to within residues 65-89 of the AMPD3 polypeptide. Ca(2+)-CaM directly stimulates isoform E catalytic activity through a K(mapp) effect and also antagonizes the protein-lipid interaction between this enzyme and intracellular membranes that inhibits catalytic activity. AMP is the predominant purine catabolite in erythrocytes deprived of glucose or exposed to A23187 ionophore alone, whereas IMP accumulates when Ca(2+) is included under the latter conditions and also during autoincubation at 37 degrees C. Preincubation with a CaM antagonist significantly slows the accumulation of erythrocyte IMP under both conditions. The combined results reveal a protein-protein interaction between Ca(2+)-CaM and isoform E and identify a mechanism that advances our understanding of erythrocyte purine metabolism. Ca(2+)-CaM overcomes potent isoform E inhibitory mechanisms that function to maintain the total adenine nucleotide pool in mature erythrocytes, which are unable to synthesize AMP from IMP because of a developmental loss of adenylosuccinate synthetase. This may also explain why Tarui's disease erythrocytes exhibit accelerated adenine nucleotide depletion in response to an increase in intracellular Ca(2+) concentration. This regulatory mechanism could also play an important role in purine metabolism in other human tissues and cells where the AMPD3 gene is expressed.

Crystallization and preliminary X-ray crystallographic analysis of adenosine 5'-monophosphate deaminase (AMPD) from Arabidopsis thaliana in complex with coformycin 5'-phosphate.

Adenosine 5'-monophosphate deaminase (AMPD) is a eukaryotic enzyme that converts adenosine 5'-monophosphate (AMP) to inosine 5'-monophosphate (IMP) and ammonia. AMPD from Arabidopsis thaliana (AtAMPD) was cloned into the baculoviral transfer vector p2Bac and co-transfected along with a modified baculoviral genome into Spodoptera frugiperda (Sf9) cells. The resulting recombinant baculovirus were plaque-purified, amplified and used to overexpress recombinant AtAMPD. Crystals of purified AtAMPD have been obtained to which coformycin 5'-phosphate, a transition-state inhibitor, is bound. Crystals belong to space group P6(2)22, with unit-cell parameters a = b = 131.325, c = 208.254 A, alpha = beta = 90, gamma = 120 degrees. Diffraction data were collected to 3.34 A resolution from a crystal in complex with coformycin 5'-phosphate and to 4.05 A resolution from a crystal of a mercury derivative.

Comparative immunologic and kinetic evaluation of AMP-deaminase isolated from normal human liver and hepatocellular carcinoma (HCC).

In the present paper physico-chemical properties of AMP-deaminase purified from human liver neoplasm-hepatocellular carcinoma (HCC) were investigated and compared with these obtained for the enzyme from normal, unaffected tissue.

AMP-deaminase from normal and cirrhotic human liver: a comparative study.

AMP-deaminase (EC 3.5.4.6) is an enzyme of nucleotide breakdown involved in regulation of adenine nucleotide pool in mammalian cells. Reaction catalysed by AMP-deaminase constitutes a rate-limiting step in adenine nucleotide catabolism in liver. In this study kinetic and regulatory properties of AMP-deaminase purified from normal and cirrhotic human liver were investigated. In comparison to AMP-deaminase extracted from the normal human liver, AMP-deaminase extracted from the cirrhotic liver was less sensitive towards substrate analogues, and only a very limited response towards pH and adenylate energy charge changes tested for enzyme isolated from this tissue source had been observed. At physiological pH 7.0, in the absence and in the presence of important allosteric effectors (ATP, ADP, GTP and orthophosphate), AMP-deaminases from the two sources studied manifested different regulatory profiles, with half-saturation constant (S0.5) values being distinctly higher for the enzyme extracted from the pathological organ. In contrast to AMP-deaminase isolated from the normal, healthy liver, where presence of relatively large (68 kDa) protein fragment was also detected, only smaller protein fragments were identified, while SDS-PAG electrophoresis of AMP-deaminase isolated from the cirrhotic liver was performed. The obtained results indicate clearly that advanced proteolytic processes occurring in the cirrhotic liver may affect structural integrity of AMP-deaminase studied, making enzyme less active and less sensitive to regulatory action of important allosteric effectors.