Use of the pyrophosphatase activity as a reliable tonoplast marker in maize roots.

Membranes of maize (Zea mays L., cv LG 11) roots were fractionated by sucrose (in presence or absence of Mg2+) or dextran density gradient centrifugations and the locations of organelles were determined using marker enzymes. Latent UDPase was used as a Golgi marker, catalase for the peroxysomes, cytochrome c oxidase for the mitochondria, UDP-Gal-galactosyltransferase for the amyloplast membranes and NADH-cytochrome c reductase for the ER. Two markers were selected for the plasmalemma, the vanadate-sensitive ATPase and UDP-Glc-sterolglucosyltransferase. The distributions of the PPase and vacuolar ATPase were found to be similar after density gradient centrifugation. The PPase and vacuolar ATPase activities were clearly separated from almost all the other markers tested, however, a partial association of both activities with the ER cannot be completely ruled out. The PPase of maize roots is more active and easier to measure than the vacuolar ATPase and is therefore an excellent candidate for use as a tonoplast marker.

Active transport of proton and calcium in higher plant cells.

Membrane transport of proton and calcium (Ca2+) plays a fundamental role in growth and developmental processes in higher plant cells. The plasma membrane contains an ATPase (P-ATPase) that pumps protons into the extracellular space, whereas two proton pumps, a vacuolar-type ATPase (V-ATPase) and a pyrophosphatase (H+-PPase) are associated with the tonoplast and pump protons into the vacuole. The P-ATPase, V-ATPase and H+-PPase catalyse electrogenic H+-translocation, giving rise to a proton motive force used to transport different molecules, via specific transport proteins (channels or carriers: H+-symport or H+-antiport), across the plasma membrane and the tonoplast

Purification and functional reconstitution of the tonoplast pyrophosphate-dependent proton pump fron Rubus hispidus cell cultures.

The hydrolytic subunit of the H+-translocating inorganic pyrophosphatase (V-PPase EC 3.6.1.1.) prepared from Rubus hispidus cell cultures has been purified from tonoplast-enriched membranes and analysed by SDS-polyacrylamide gel electrophoresis, Only one polypeptide of M(r) 70 000 was recovered with the V-PPase activity after solubilization in the presence of Triton X-100, purification by gel filtration (Superose) and anion exchange (Mono Q) chromatography. This polypeptide strongly cross-reacted with an antibody raised against the V-PPase from Vigna radiata. The tonoplast-enriched fraction was also used to solubilize and reconstitute the-V-PPase. The proteoliposomes showing a PPi-dependent proton transport activity were purified by gel filtration (Superose) and analysed by SDS-polyacrylamide gel electrophoresis. Only one polypeptide of M(r) 70 000 was recovered with the proton-pumping activity. All these data suggest that the native V-PPase from Rubus is composed of a single kind of polypeptide with an M(r) of 70 000 and representing the catalytic subunit.

Target Molecular Size and Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Analysis of the ATP-and Pyrophosphate-Dependent Proton Pumps from Maize Root Tonoplast.

Tonoplast-enriched membranes were prepared from maize (Zea mays L. cv LG 11) primary roots, using sucrose nonlinear gradients. The functional molecular size of the tonoplast ATP-and PPi-dependent proton pumps were analyzed by radiation inactivation. Glucose-6-phosphate dehydrogenase (G6PDH) was added as an internal standard. Frozen samples (-196 degrees C) of the membranes were irradiated with (60)Co for different periods of time. After thawing the samples, the activities of G6PDH, ATPase, and PPase were tested. By applying target theory, the functional sizes of the ATPase and PPase in situ were found to be around 540 and 160 kilodaltons, respectively. The two activities were solubilized and separated by gel filtration chromatography. The different polypeptides copurifying with the two pumps were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two bands (around 59 and 65 kilodaltons) were associated with the ATPase activity, whereas a double band (around 40 kilodaltons) was recovered with the PPase activity.

Atividade ATPásica e pirofosfatásica em microssomos de raízes de milho colonizadas com fungos micorrízicos arbusculares

No presente trabalho, foi avaliada a influência de duas espécies de fungos micorrízicos arbusculares (FMAs) Glomus clarum e Gigaspora margarita sobre as atividades ATPásica e pirofosfatásica de microssomos obtidos por meio de fracionamento celular de raízes de milho colonizadas aos 20, 30, 40 e 60 dias do plantio. Ambos os fungos proporcionaram aumentos significativos nas atividades das ATPases e pirofosfatases; entretanto, as maiores atividades foram observadas nas raízes colonizadas pelo fungo G. clarum. Os dados cinéticos indicaram a presença de uma ativação diferencial das H+-ATPases e H+-pirofosfatases presentes nas membranas das células radiculares, dependendo da espécie fúngica e do estádio da colonização das raízes pelos FMAs. Como indicadores da eficiência da micorrização, foram avaliados a altura e o conteúdo de nutrientes na parte aérea das plantas. Os dados obtidos mostraram a primeira evidência cinética de estimulação de atividade pirofosfatásica em membranas microssomais de raízes colonizadas e descreveram um padrão inédito de ativação diferencial para a hidrólise de ATP, dependendo da espécie (G. clarum ou G. margarita) e do estádio de colonização.

The Structural Basis for inorganic Pyrophosphatase Catalysis and Regulation

Inorganic pyrophosphatases (PPases) are essential enzymes for every living cell. PPases provide the necessary thermodynamic pull for many biosynthetic reactions by hydrolyzing pyrophosphate. There are two types of PPases: integral membrane-bound and soluble enzymes. The latter type is divided into two non-homologous protein families, I and II. Family I PPases are present in all kingdoms of life, whereas family II PPases are only found in prokaryotes, including archae. Family I PPases, particularly that from Saccharomyces cerevisiae, are among the most extensively characterized phosphoryl transfer enzymes. In the present study, we have solved the structures of wild-type and seven active site variants of S. cerevisiae PPase bound to its natural metal cofactor, magnesium ion. These structures have facilitated derivation of the complete enzyme reaction scheme for PPase, fulfilling structures of all the reaction intermediates. The main focus in this study was on a novel subfamily of family II PPases (CBSPPase) containing a large insert formed by two CBS domains and a DRTGG domain within the catalytic domain. The CBS domain (named after cystathionine beta-synthase in which it was initially identified) usually occurs as tandem pairs with two or four copies in many proteins in all kingdoms of life. The structure formed by a pair of CBS domains is also known as a Bateman domain. CBS domains function as regulatory units, with adenylate ligands as the main effectors. The DRTGG domain (designated based on its most conserved residues) occurs less frequently and only in prokaryotes. Often, the domain co-exists with CBS domains, but its function remains unknown. The key objective of the current study was to explore the structural rearrangements in the CBS domains induced by regulatory adenylate ligands and their functional consequences. Two CBS-PPases were investigated, one from Clostridium perfringens (cpCBS-PPase) containing both CBS and DRTGG domains in its regulatory region and the other from Moorella thermoacetica (mt CBS-PPase) lacking the DRTGG domain. We additionally constructed a separate regulatory region of cpCBS-PPase (cpCBS). Both full-length enzymes and cpCBS formed homodimers. Two structures of the regulatory region of cpCBS-PPase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate, were solved. The structures were significantly different, providing information on the structural pathway from bound adenylates to the interface between the regulatory and catalytic parts. To our knowledge, these are the first reported structures of a regulated CBS enzyme, which reveal large conformational changes upon regulator binding. The activator-bound structure was more open, consistent with the different thermostabilities of the activator- and inhibitor-bound forms of cpCBS-PPase. The results of the functional studies on wild-type and variant CBS-PPases provide support for inferences made on the basis of structural analyses. Moreover, these findings indicate that CBS-PPase activity is highly sensitive to adenine nucleotide distribution between AMP, ADP and ATP, and hence to the energy level of the cell. CBS-PPase activity is markedly inhibited at low energy levels, allowing PPi energy to be used for cell survival instead of being converted into heat.

Functional studies on bacterial nucleotide-regulated inorganic pyrophosphatases

CBS domains are ~60 amino acid tandemly repeated regulatory modules forming a widely distributed domain superfamily. Found in thousands of proteins from all kingdoms of life, CBS domains have adopted a variety of functions during evolution, one of which is regulation of enzyme activity through binding of adenylate-containing compounds in a hydrophobic cavity. Mutations in human CBS domain-containing proteins cause hereditary diseases. Inorganic pyrophosphatases (PPases) are ubiquitous enzymes, which pull pyrophosphate (PPi) producing reactions forward by hydrolyzing PPi into phosphate. Of the two nonhomologous soluble PPases, dimeric family II PPases, belonging to the DHH family of phosphoesterases, require a transition metal and magnesium for maximal activity. A quarter of the almost 500 family II PPases, found in bacteria and archaea, contain a 120-250 amino acid N-terminal insertion, comprised of two CBS domains separated in sequence by a DRTGG domain. These enzymes are thus named CBS-PPases. The function of the DRTGG domain in proteins is unknown. The aim of this PhD thesis was to elucidate the structural and functional differences of CBS-PPases in comparison to family II PPases lacking the regulatory insert. To this end, we expressed, purified and characterized the CBS-PPases from Clostridium perfringens (cpCBS-PPase) and Moorella thermoacetica (mtCBS-PPase), the latter lacking a DRTGG domain. Both enzymes are homodimers in solution and display maximal activity against PPi in the presence of Co2+ and Mg2+. Uniquely, the DRTGG domain was found to enable tripolyphosphate hydrolysis at rates similar to that of PPi. Additionally, we found that AMP and ADP inhibit, while ATP and AP4A activate CBSPPases, thus enabling regulation in response to changes in cellular energy status. We then observed substrate- and nucleotide-induced conformational transitions in mtCBS-PPase and found that the enzyme exists in two differentially active conformations, interconverted through substrate binding and resulting in a 2.5-fold enzyme activation. AMP binding was shown to produce an alternate conformation, which is reached through a different pathway than the substrate-induced conformation. We solved the structure of the regulatory insert from cpCBS-PPase in complex with AMP and AP4A and proposed that conformational changes in the loops connecting the catalytic and regulatory domains enable activity regulation. We examined the effects of mutations in the CBS domains of mtCBS-PPase on catalytic activity, as well as, nucleotide binding and inhibition.

Sistemas primários de transporte de prótons integram os mecanismos de desintoxicação do mesotrione em plantas de milho

O mesotrione é um dos mais efetivos herbicidas desenvolvidos para o controle de uma ampla gama de plantas daninhas que infestam campos de milho (Zea mays). Todavia, as bases bioquímicas e moleculares da tolerância das plantas de milho a esse herbicida ainda não foram estabelecidas. Para compreender os mecanismos de desintoxicação do mesotrione em plantas de milho, foram analisadas as atividades dos principais sistemas primários de transporte de prótons (íons H+) das membranas plasmática e vacuolar (H+-ATPases do tipo P e V e H+-PPases) de células de diferentes tecidos de plantas tratadas após aplicação do herbicida em pós-emergência. Para isso, foram realizados procedimentos de fracionamento celular, de tecidos radiculares, foliares e do caule, por centrifugação diferencial e purificação de vesículas membranares em gradiente de densidade de sacarose. Os ensaios enzimáticos das atividades hidrolíticas das três bombas de H+ foram realizados aplicando-se um método colorimétrico para medir o fosfato liberado das hidrólises dos substratos: adenosina-5'-trifosfato (ATP) e pirofosfato (PPi). Parâmetros fotossintéticos foram analisados como marcadores fisiológicos dos diferentes estádios da desintoxicação das plantas. Essa análise demonstrou que o tratamento com mesotrione promoveu uma redução na taxa fotossintética e na relação Fv/Fm no terceiro dia após aplicação (DAA), mas não afetou significativamente a fotossíntese a partir do quinto DAA. Nos três tecidos analisados, raiz, folha e caule, aos 3 DAA, foi observado forte estímulo da atividade da H+-PPase vacuolar, a qual variou de cerca de 100 a 600%. Essa forte ativação foi reduzida significativamente aos 7 DAA, mas permaneceu pelo menos duas vezes maior com relação ao controle. Por sua vez, as H+-ATPases das membranas plasmática e vacuolar foram bem menos moduladas pelo tratamento com o herbicida, apresentando estimulações e inibições que não variaram mais do que 20 a 60% das atividades obtidas em vesículas de membranas oriundas de plantas não tratadas (controle). Os resultados demonstraram que o mesotrione promove uma ativação diferencial dos principais sistemas primários de transporte de H+, indicando que essas bombas iônicas são enzimas transportadoras essenciais aos mecanismos relacionados com o processo de desintoxicação das plantas de milho, possivelmente ao energizar a compartimentalização das moléculas do herbicida mesotrione no vacúolo ou a exceção celular através das membranas plasmáticas.

Unraveling the functional divergence of membrane-bound pyrophosphatases

Inorganic pyrophosphatases (PPases) are enzymes that hydrolyze pyrophosphate (PPi)which is produced as a byproduct in many important growth related processes e.g. in the biosynthesis of DNA, proteins and lipids. PPases can be either soluble or membranebound. Membrane-bound PPases (mPPases) are ion transporters that couple the energy released during PPi hydrolysis to Na+ or H+ transport. When I started the project, only three Na+-transporting mPPases were known to exist. In this study, I aimed to confirm if Na+-transport is a common function of mPPases. Furthermore, the amino acid residues responsible for determining the transporter specificity were unknown. I constructed a phylogenetic tree for mPPases and selected the representative bacterial and archaeal mPPases to be investigated. I expressed different prokaryotic mPPases in Escherichia coli, isolated these as inverted membrane vesicles and characterized their functions. In the first project I identified four new Na+-PPases, two K+-dependent H+-PPases and one K+-independent mPPase. The residues determining the transporter specificity were identified by site-directed mutagenesis. I showed that the conserved glutamate residues are important for specificity, though are not the only residues that influence it. This research clarified the ion transport specificities throughout the mPPase phylogenetic tree, and revealed that Na+ transport is a widespread function of mPPases. In addition, it became clear that the transporter specificity can be predicted from the amino acid sequence in combination with a phylogenetic analysis. In the second project, I identified a novel class of mPPases, which is capable of transporting both Na+ and H+ ions and is mainly found in bacteria of the human gastrointestinal tract. The physiological role of these novel enzymes may be to help the bacteria survive in the demanding conditions of the host. In the third project, I characterized the Chlorobium limicola Na+-PPase and found that this and related mPPases are able to transport H+ ions at subphysiological Na+ concentrations. In addition, the H+-transport activity was shown to be a common function of all studied Na+-PPases at low Na+ concentrations. I observed that mutating gate-lysine to asparagine eliminated the H+ but not the Na+ ion transport function, indicating the important role of the residue in the transport of H+. In the fourth project, I characterized the unknown and evolutionary divergent mPPase clade of the phylogenetic tree. The enzymes belonging to this clade are able to transport H+ ions and, based on their sequence, were expected to be K+- and Na+-independent. The sequences of membrane-bound PPase are usually highly conserved, but the enzymes belonging to this clade are more divergent and usually contain 100−150 extra amino acid residues compared to other known mPPases. Despite the vast sequence differences, these mPPases have the full set of important residues and, surprisingly, are regulated by Na+ and K+ ions. These enzymes are mainly of bacterial origin.

Sucrose and nitrogen supplies regulate growth of maize kernels

The growth of maize (Zea mays L.) kernels depends on the availability of carbon (C) and nitrogen (N) assimilates supplied by the mother plant and the capacity of the kernel to use them. Our objectives were to study the effects of N and sucrose supply levels on growth and metabolism of maize kernels. Kernel explants of Pioneer 34RO6 were cultured in vitro with varying combinations of N (5 to 30 mM) and sucrose (117 to 467 mM). Maximum kernel growth was obtained with 10 mM N and 292 mM sucrose in the medium, and a deficiency of one assimilate could not be overcome by a sufficiency of the other. Increasing the N supply led to increases in the kernel sink capacity (number of cells and starch granules in the endosperm), activity of certain enzymes (soluble and bound invertases, sucrose synthase, and aspartate aminotransaminase), starch, and the levels of N compounds (total-N, soluble protein, and free amino acids), and decreased the levels of C metabolites (sucrose and reducing sugars). Conversely, increasing the sucrose supply increased the level of endosperm C metabolites, free amino acids, and ADPG-PPase and alanine transaminase activities, but decreased the activity of soluble invertase and concentrations of soluble protein and total-N. Thus, while C and N are interdependent and essential for accumulation of maximum kernel weight, they appear to regulate growth by different means. Nitrogen supply aids the establishment of kernel sink capacity, and promotes activity of enzymes relating to sucrose and nitrogen uptake, while sucrose regulates the activities df invertase and ADPG-PPase. (C) 1999 Annals of Botany Company.

Análise in silico de uma pirofosfatase e de uma tiosterase de uma biblioteca metagenômica de solo de Eucalipto spp

Pós-graduação em Microbiologia Agropecuária - FCAV

Novel inorganic pyrophosphatase from soil metagenomic and family and subfamily prediction

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

MetaPPase by in silico analyses predicting the novel gene operate as H+ pumps.

The lastyears declined the discovery of compounds to use in industrial and naturaldiversity has been the best supplier for novel genes, enzymes and compounds inhigh demand by the biotechnology industry. We know immense diversity of microorganisms,yet most remains unexplored. For these reason we use the metagenômica approach toinvestigate the potential of uncultured microorganisms. With this purpose weused the metagenomic library of from Eucalyptus spp. arboretum (EAA), wedid screening to found positive clone and them was submitted to the process of shotgun,the data obtained was submitted a bioinformatics analyses. Our results showsthe hypothesis of high unexplored microbial diversity of soil are able to foundnovel genes and metagenomic approach is and allowed to isolate novel genes and insilico analyses are essential part to identify a novel Inorganicpyrophosphatase (PPase) prediction indicated the novel gene operate as H+ pumps. Thissuggests that a special feature, our work in situ will be cloning thegene expression vector for subsequent kinetic characterization and crystallization.

Toward a quantum-mechanical description of metal-assisted phosphoryl transfer in pyrophosphatase

The wealth of kinetic and structural information makes inorganic pyrophosphatases (PPases) a good model system to study the details of enzymatic phosphoryl transfer. The enzyme accelerates metal-complexed phosphoryl transfer 1010-fold: but how? Our structures of the yeast PPase product complex at 1.15 Å and fluoride-inhibited complex at 1.9 Å visualize the active site in three different states: substrate-bound, immediate product bound, and relaxed product bound. These span the steps around chemical catalysis and provide strong evidence that a water molecule (Onu) directly attacks PPi with a pKa vastly lowered by coordination to two metal ions and D117. They also suggest that a low-barrier hydrogen bond (LBHB) forms between D117 and Onu, in part because of steric crowding by W100 and N116. Direct visualization of the double bonds on the phosphates appears possible. The flexible side chains at the top of the active site absorb the motion involved in the reaction, which may help accelerate catalysis. Relaxation of the product allows a new nucleophile to be generated and creates symmetry in the elementary catalytic steps on the enzyme. We are thus moving closer to understanding phosphoryl transfer in PPases at the quantum mechanical level. Ultra-high resolution structures can thus tease out overlapping complexes and so are as relevant to discussion of enzyme mechanism as structures produced by time-resolved crystallography.

Mutagenesis of the Glucose-1-Phosphate-Binding Site of Potato Tuber ADP-Glucose Pyrophosphorylase1

Lysine (Lys)-195 in the homotetrameric ADP-glucose pyrophosphorylase (ADPGlc PPase) from Escherichia coli was shown previously to be involved in the binding of the substrate glucose-1-phosphate (Glc-1-P). This residue is highly conserved in the ADPGlc PPase family. Site-directed mutagenesis was used to investigate the function of this conserved Lys residue in the large and small subunits of the heterotetrameric potato (Solanum tuberosum) tuber enzyme. The apparent affinity for Glc-1-P of the wild-type enzyme decreased 135- to 550-fold by changing Lys-198 of the small subunit to arginine, alanine, or glutamic acid, suggesting that both the charge and the size of this residue influence Glc-1-P binding. These mutations had little effect on the kinetic constants for the other substrates (ATP and Mg2+ or ADP-Glc and inorganic phosphate), activator (3-phosphoglycerate), inhibitor (inorganic phosphate), or on the thermal stability. Mutagenesis of the corresponding Lys (Lys-213) in the large subunit had no effect on the apparent affinity for Glc-1-P by substitution with arginine, alanine, or glutamic acid. A double mutant, SK198RLK213R, was also obtained that had a 100-fold reduction of the apparent affinity for Glc-1-P. The data indicate that Lys-198 in the small subunit is directly involved in the binding of Glc-1-P, whereas they appear to exclude a direct role of Lys-213 in the large subunit in the interaction with this substrate.