Letteratura scientifica selezionata sul tema "Pyruvate decarboxylase"

S-Adenosylmethionine decarboxylase is a key enzyme for the synthesis of polyamines in mammals, plants and many other species that use aminopropyltransferases for this pathway. It catalyses the formation of S-adenosyl-1-(methylthio)-3-propylamine (decarboxylated S-adenosylmethionine), which is used as the aminopropyl donor. This is the sole function of decarboxylated S-adenosylmethionine. Its content is therefore kept very low and is regulated by variation in the activity of S-adenosylmethionine decarboxylase according to the need for polyamine synthesis. All S-adenosylmethionine decarboxylases have a covalently bound pyruvate prosthetic group, which is essential for the decarboxylation reaction, and have similar structures, although they differ with respect to activation by cations, primary sequence and subunit composition. The present chapter describes these features, the mechanisms for autocatalytic generation of the pyruvate from a proenzyme precursor and for the decarboxylation reaction, and the available inhibitors of this enzyme, which have uses as anticancer and anti-trypanosomal agents. The intricate mechanisms for regulation of mammalian S-adenosylmethionine decarboxylase activity and content are also described.

ABSTRACT The fusel alcohols 3-methyl-1-butanol, 2-methyl-1-butanol, and 2-methyl-propanol are important flavor compounds in yeast-derived food products and beverages. The formation of these compounds from branched-chain amino acids is generally assumed to occur via the Ehrlich pathway, which involves the concerted action of a branched-chain transaminase, a decarboxylase, and an alcohol dehydrogenase. Partially purified preparations of pyruvate decarboxylase (EC 4.1.1.1 ) have been reported to catalyze the decarboxylation of the branched-chain 2-oxo acids formed upon transamination of leucine, isoleucine, and valine. Indeed, in a coupled enzymatic assay with horse liver alcohol dehydrogenase, cell extracts of a wild-type Saccharomyces cerevisiae strain exhibited significant decarboxylation rates with these branched-chain 2-oxo acids. Decarboxylation of branched-chain 2-oxo acids was not detectable in cell extracts of an isogenic strain in which all threePDC genes had been disrupted. Experiments with cell extracts from S. cerevisiae mutants expressing a singlePDC gene demonstrated that both PDC1- andPDC5-encoded isoenzymes can decarboxylate branched-chain 2-oxo acids. To investigate whether pyruvate decarboxylase is essential for fusel alcohol production by whole cells, wild-type S. cerevisiae and an isogenic pyruvate decarboxylase-negative strain were grown on ethanol with a mixture of leucine, isoleucine, and valine as the nitrogen source. Surprisingly, the three corresponding fusel alcohols were produced in both strains. This result proves that decarboxylation of branched-chain 2-oxo acids via pyruvate decarboxylase is not an essential step in fusel alcohol production.

Oxythiamine is an antivitamin derivative of thiamine that after phosphorylation to oxythiamine pyro phos phate can bind to the active centres of thiamine-dependent enzymes. In the present study, the effect of oxythiamine on the viability of Saccharomyces cerevisiae and the activity of thiamine pyrophosphate dependent enzymes in yeast cells has been investigated. We observed a decrease in pyruvate decarboxylase specific activity on both a control and an oxythiamine medium after the first 6 h of culture. The cytosolic enzymes transketolase and pyruvate decarboxylase decreased their specific activity in the presence of oxythiamine but only during the beginning of the cultivation. However, after 12 h of cultivation, oxythiamine-treated cells showed higher specific activity of cytosolic enzymes. More over, it was established by SDS–PAGE that the high specific activity of pyruvate decarboxylase was followed by an increase in the amount of the enzyme protein. In contrast, the mitochondrial enzymes, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes, were inhibited by oxythiamine during the entire experiment. Our results suggest that the observed strong decrease in growth rate and viability of yeast on medium with oxythiamine may be due to stronger in hibition of mitochondrial pyruvate dehydrogenase than of cytosolic enzymes.Key words: pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, transketolase, pyruvate decarboxylase, activity, oxythiamine, inhibition.

Clostridium acetobutylicum ATCC 824 using pyruvate as the sole carbon source produced mainly acetate and butyrate as end products of fermentation. Acetate and butyrate kinase activities were higher in cells growing in the presence of pyruvate than glucose, whereas the level of the acetoacetate decarboxylase, an enzyme involved in solvent formation, was lower. Similar activities of glyceraldehyde-3-phosphate dehydrogenase were found in cells grown in pyruvate and glucose mediums. The transfer of C. acetobutylicum from pyruvate to glucose medium suggested that pyruvate represses the "solventogenesis."Key words: Clostridium acetobutylicum, pyruvic acid, kinase, acetoacetate decarboxylase.

ABSTRACTFusel alcohols are precursors and contributors to flavor and aroma compounds in fermented beverages, and some are under investigation as biofuels. The decarboxylation of 2-oxo acids is a key step in the Ehrlich pathway for fusel alcohol production. InSaccharomyces cerevisiae, five genes share sequence similarity with genes encoding thiamine pyrophosphate-dependent 2-oxo-acid decarboxylases (2ODCs).PDC1,PDC5, andPDC6encode differentially regulated pyruvate decarboxylase isoenzymes;ARO10encodes a 2-oxo-acid decarboxylase with broad substrate specificity, andTHI3has not yet been shown to encode an active decarboxylase. Despite the importance of fusel alcohol production inS. cerevisiae, the substrate specificities of these five 2ODCs have not been systematically compared. When the five 2ODCs were individually overexpressed in apdc1Δpdc5Δpdc6Δaro10Δthi3Δ strain, only Pdc1, Pdc5, and Pdc6 catalyzed the decarboxylation of the linear-chain 2-oxo acids pyruvate, 2-oxo-butanoate, and 2-oxo-pentanoate in cell extracts. The presence of a Pdc isoenzyme was also required for the production ofn-propanol andn-butanol in cultures grown on threonine and norvaline, respectively, as nitrogen sources. These results demonstrate the importance of pyruvate decarboxylases in the natural production ofn-propanol andn-butanol byS. cerevisiae. No decarboxylation activity was found for Thi3 with any of the substrates tested. Only Aro10 and Pdc5 catalyzed the decarboxylation of the aromatic substrate phenylpyruvate, with Aro10 showing superior kinetic properties. Aro10, Pdc1, Pdc5, and Pdc6 exhibited activity with all branched-chain and sulfur-containing 2-oxo acids tested but with markedly different decarboxylation kinetics. The high affinity of Aro10 identified it as a key contributor to the production of branched-chain and sulfur-containing fusel alcohols.

The hyperthermophilic archaeonThermococcus guaymasensisproduces ethanol as a metabolic end product, and an alcohol dehydrogenase (ADH) catalyzing the reduction of acetaldehyde to ethanol has been purified and characterized. However, the enzyme catalyzing the formation of acetaldehyde has not been identified. In this study an enzyme catalyzing the production of acetaldehyde from pyruvate was purified and characterized fromT. guaymasensisunder strictly anaerobic conditions. The enzyme had both pyruvate decarboxylase (PDC) and pyruvate ferredoxin oxidoreductase (POR) activities. It was oxygen sensitive, and the optimal temperatures were 85°C and >95°C for the PDC and POR activities, respectively. The purified enzyme had activities of3.8±0.22 U mg−1and20.2±1.8 U mg−1, with optimal pH-values of 9.5 and 8.4 for each activity, respectively. Coenzyme A was essential for both activities, although it did not serve as a substrate for the former. Enzyme kinetic parameters were determined separately for each activity. The purified enzyme was a heterotetramer. The sequences of the genes encoding the subunits of the bifunctional PDC/POR were determined. It is predicted that all hyperthermophilicβ-keto acids ferredoxin oxidoreductases are bifunctional, catalyzing the activities of nonoxidative and oxidative decarboxylation of the correspondingβ-keto acids.

ABSTRACTA pathway toward isobutanol production previously constructed inEscherichia coliinvolves 2-ketoacid decarboxylase (Kdc) fromLactococcus lactisthat decarboxylates 2-ketoisovalerate (KIV) to isobutyraldehyde. Here, we showed that a strain lacking Kdc is still capable of producing isobutanol. We found that acetolactate synthase fromBacillus subtilis(AlsS), which originally catalyzes the condensation of two molecules of pyruvate to form 2-acetolactate, is able to catalyze the decarboxylation of KIV like Kdc both in vivo and in vitro. Mutational studies revealed that the replacement of Q487 with amino acids with small side chains (Ala, Ser, and Gly) diminished only the decarboxylase activity but maintained the synthase activity.

(2S)- and (2R)-Serine O-sulphate have been synthesised and shown to inactivate glutamate decarboxylase (GAD) from E. Coli. Novel methodology was developed to enable the stereospecific synthesis of (2S) and (2R)-deuteriated serine in order to probe the mechanism of inactivation. The rates of inactivation of glutamate decarboxylase by (2S)-, (2S)-[2-2H]-, (2R)- and (2R)-[2-2H]-serine O-sulphate have been measured for each of the isotopomers at a range of concentrations. From the data obtained the deuterium isotope effects were determined for each enantiomer. The inactivation by the (2S)-enantiomer was shown to involve C-H bond cleavage while inactivation by the (2R)-isomer involves C-decarboxylation. Both processes were shown to occur on the 4'-re-face of the coenzyme, the opposite face to that utilised in the physiological decarboxylation reaction. The methodology developed for the synthesis of the deuteriated serines involved the regiospecific introduction of deuterium to the C-6 centre of (3R)- and (3S)-2,5- dimethoxy-3-isopropyl-3,6-dihydropyrazine. Schollkopf chemistry was then exploited for the stereospecific alkylation at C-6 of the dihydropyrazines. This chemistry was versatile and enabled the synthesis of other deuteriated amino acids. For example (2S)-[2-2H]-phenylalanine, (2S)-[2-2H]-allylglycine and (2S)-[2-2H]-aspartic acid were synthesised using this chemistry. The decarboxylation of 2-aminomalonic acid by cytosolic serine hydroxymethyltransferase (SHMT) was studied. Contrary to previous reports, the reaction was found to be stereospecific and the newly introduced hydrogen was shown to occupy the 2-pro-S position of the glycine product.

Bioethanol, produced from organic waste as a second-generation biofuel, is an important renewable energy source. Here, recalcitrant carbohydrate sources, such as municipal and agricultural waste, and plants grown on land not suitable for food crops, are exploited. The thermophilic, Gram-positive bacterium Geobacillus thermoglucosidasius is naturally very flexible in its growth substrates and produces a variety of fermentation products, including lactate, formate, acetate and ethanol. TMO Renewables Ltd. used metabolic engineering to enhance ethanol production, creating the production strain TM242 (NCIMB 11955 ∆ldh, ∆pfl, pdhup). Ethanol yield has been increased to 82% of the theoretical maximum on glucose and up to 92% of the theoretical maximum on cellobiose. However, this strain still produces acetate, presumably derived from the overproduction of acetyl-CoA through the upregulated pdh gene encoding the pyruvate dehydrogenase complex. An alternative to the mixed fermentation pathway found in G. thermoglucosidasius is to introduce a homoethanologenic pathway. Yeast and a very limited range of mesophilic bacteria use the homoethanol fermentation pathway, which employs pyruvate decarboxylase (PDC) in conjunction with alcohol dehydrogenase (ADH), to convert pyruvate to ethanol. Despite extensive screening, no PDC has yet been identified in a thermophilic organism. Using the thermophile G. thermoglucosidasius as a host platform, we endeavoured to develop a thermophilic version of the homoethanol pathway for use in Geobacillus spp. This Thesis reports the in vitro characterization and crystal structure of one of the most thermostable bacterial PDCs from the mesophile Zymobacter palmae (ZpPDC) and describes strategies to improve expression of active PDC at high growth temperatures. This includes codon harmonization and the successful development of a PET (producer of ethanol) operon. Furthermore, ancestral sequence reconstruction was explored as an alternative engineering approach, but did not yield a PDC more thermostable than ZpPDC. In vitro ZpPDC is most active at 65°C with a denaturation temperature of 70°C, when sourced from a recombinant mesophilic host. Codon harmonization improved detectable PDC activity in G. thermoglucosidasius cultures grown up to 65°C by up to 42%. Pairing this PDC with G. thermoglucosidasius ADH6 produced a PET functional up to 65°C with ethanol yields of 87% of the theoretical maximum on glucose. This increase in yield at temperatures of up to 15°C higher than previously reported for any PDC expressed.

La present tesi es centra en el desenvolupament i optimització d’una estratègia basada en la biocatàlisi per a la síntesi d’amines quirals, les quals són compostos òpticament actius de gran valor que poden ésser utilitzats per a la síntesi de nombrosos productes, especialment en les indústries farmacèutica i agroquímica. Més concretament, es pretén sintetitzar 3-amino-1-fenilbutà (3-APB) i 1-feniletilamina (1-PEA) a través de la reacció en cascada de la transaminasa (TA) i la piruvat decarboxilasa (PDC). Aquesta cascada es basa en una síntesi asimètrica que parteix de les seves corresponents cetones proquirals i l’alanina, i és catalitzada per omega-transaminases, les quals presenten un equilibri desfavorable. Per tal de solucionar aquest problema, la PDC actua com un sistema d’eliminació de producte secundari, a través de la transformació del piruvat a acetaldehid i CO2, la qual cosa provoca un desplaçament de l’equilibri. Amb l’objectiu de superar les limitacions comercials de la PDC, la qual només es pot obtenir en petites quantitats a un cost alt, es va desenvolupar un procés sencer de producció d’aquest enzim. Es va clonar i sobreexpressar el gen de la PDC de Zymobacter palmae (ZpPDC) en Escherichia coli. Posteriorment, es va obtenir l’enzim recombinant en grans quantitats a través del desenvolupament d’un procés de cultiu d’alta densitat cel·lular en bioreactor. Pel que fa a les TAs, es disposava de quatre enzims diferents, procedents de Chromobacterium violaceum (Cvi-TA), Vibrio fluvialis (Vfl-TA) i Aspergillus terreus (Ate-TA i Ate-TA_T247S). Es va caracteritzar tant la PDC com les quatre transaminases per tal de trobar les condicions de compromís adequades per a la construcció de la cascada enzimàtica. Tenint en compte les condicions trobades, es va dur a terme, de forma preliminar, reaccions de cribratge de les quals en van sortir seleccionades la Cvi-TA i la Vfl-TA per a la síntesi de 3-APB; i Vfl-TA per a la síntesi de 1-PEA. Després de demostrar la viabilitat de la reacció en cascada de la TA i la PDC, es van aplicar diferents estratègies d’optimització per tal de maximitzar els rendiments de reacció i millorar la baixa estabilitat operacional de les transaminases. Per una banda, es van explorar algunes estratègies d’optimització de les condicions de reacció. Per l’altra, es va aplicar enginyeria del medi de reacció. Posteriorment, es va dur a terme d’immobilització dels enzims. Es van obtenir derivats immobilitzats tant de la Cvi-TA com de la Vfl-TA en suports de MANA-agarosa i epoxy-agarosa. En el cas de la PDC, es va desenvolupar un sistema innovador de purificació i immobilització simultània en MANA-agarosa. Finalment, els enzims immobilitzats obtinguts van ser aplicats en reacció i es va desenvolupar una estratègia de reacció en cicles.
La presente tesis se centra en el desarrollo y optimización de una estrategia basada en la biocatálisis para la síntesis de aminas quirales, las cuales son compuestos ópticamente activos de gran valor que pueden ser utilizados para la síntesis de numerosos productos, especialmente en las industrias farmacéutica y agroquímica. Más concretamente, se pretende sintetizar 3-amino-1-fenilbutano (3-APB) y 1-feniletilamina (1-PEA) a través de la reacción en cascada de la transaminasa (TA) y la piruvato decarboxilasa (PDC). Esta cascada se basa en una síntesis asimétrica que parte de sus correspondientes cetonas proquirales y la alanina, y es catalizada por omega-transaminasas, las que presentan un equilibrio desfavorable. Para solucionar este problema, la PDC actúa como un sistema de eliminación de producto secundario, a través de la transformación del piruvato en acetaldehído y CO2, lo que provoca un desplazamiento del equilibrio. Con el objetivo de superar las limitaciones comerciales de la PDC, la cual sólo se puede obtener en pequeñas cantidades a un coste alto, se desarrolló un proceso entero de producción de esta enzima. Se clonó y sobreexpresó el gen de la PDC de Zymobacter Palmae (ZpPDC) en Escherichia coli. Posteriormente, se obtuvo la enzima recombinante en grandes cantidades a través del desarrollo de un proceso de cultivo de alta densidad celular en bioreactor. En cuanto a las TAs, se disponía de cuatro enzimas diferentes, procedentes de Chromobacterium violaceum (Cvi-TA), Vibrio fluvial (Vfl-TA) y Aspergillus Terreus (Ate-TA y Ate-TA_T247S). Se caracterizó tanto la PDC como las cuatro transaminasas con el fin de encontrar las condiciones de compromiso adecuadas para la construcción de la cascada enzimática. Teniendo en cuenta las condiciones encontradas, se llevó a cabo, de forma preliminar, reacciones de cribado de las que salieron seleccionadas la Cvi-TA y la Vfl-TA para la síntesis de 3-APB; y Vfl-TA para la síntesis de 1-PEA. Tras demostrar la viabilidad de la reacción en cascada de la TA y la PDC, se aplicaron diferentes estrategias de optimización para maximizar los rendimientos de reacción y mejorar la baja estabilidad operacional de las transaminasas. Por un lado, se exploraron algunas estrategias de optimización de las condiciones de reacción. Por el otro, se aplicó ingeniería del medio de reacción. Posteriormente, se llevó a cabo de inmovilización de las enzimas. Se obtuvieron derivados inmovilizados tanto de la Cvi-TA como de la Vfl-TA en soportes de MANA-agarosa y epoxy-agarosa. En el caso de la PDC, se desarrolló un sistema innovador de purificación e inmovilización simultánea en MANA-agarosa. Finalmente, las enzimas inmovilizadas obtenidas fueron aplicadas en reacción y se desarrolló una estrategia de reacción en ciclos.
The present thesis is focused on the development and optimization of a biocatalytical approach for the synthesis of chiral amines, which are highly valuable optically active compounds that can be used for the synthesis of numerous targets, especially in pharmaceutical and agrochemical industry. More specifically, 3-amino-1-phenylbutane (3-APB) and 1-phenylethylamine (1-PEA) synthesis is pretended by the cascade reaction of transaminase (TA) and pyruvate decarboxylase (PDC). The mentioned cascade consists in an asymmetric synthesis from their corresponding prochiral ketones and alanine catalyzed by omega-transaminase, which presents an unfavorable equilibrium. To overcome this problem, PDC acts as a by product removing system by transforming the resulting pyruvate to acetaldehyde and CO2, which leads to an equilibrium shift. Aiming to overcome the low PDC commercial availability, which can only be acquired at low amounts and a high cost, a whole production process was developed. Zymobacter palmae PDC (ZpPDC) gene was cloned and overexpressed in Escherichia coli. After that, high amounts of the recombinant enzyme were obtained by the development of a high-cell density culture process in bench-top bioreactor. Regarding TA, four different enzymes were available from Chromobacterium violaceum (Cvi-TA), Vibrio fluvialis (Vfl-TA) and Aspergillus terreus (Ate-TA and Ate-TA_T247S). Both PDC and the different transaminases were characterized to find out the appropriate compromise conditions to construct the enzymatic cascade. Taking into account the found conditions, preliminary screening reactions were carried out, from which Cvi-TA and Vfl-TA were selected for the synthesis of 3-APB; and Vfl-TA for the synthesis of 1-PEA. After proving the feasibility of TA and PDC cascade reaction, different optimization approaches were applied in order to maximize reaction yields and to improve the low transaminase operational stability. On the one hand, reaction conditions optimization approaches were explored. On the other, reaction medium engineering was applied. After that, enzyme immobilization was carried out. Immobilized derivatives of both Cvi-TA and Vfl-TA were obtained in MANA-agarose and epoxy-agarose supports. In the case of PDC, an innovative simultaneous purification and immobilization process was developed using MANA-agarose. Finally, the obtained immobilized enzymes were applied in reactions and a reaction cycle strategy was developed.
Universitat Autònoma de Barcelona. Programa de Doctorat en Biotecnologia

The effect of surfactants on the hydrolysis of achiral and chiral substrates by crude and purified porcine pancreatic lipase (PPL; EC 3.1.1.3) has been studied. Rather than accelerating the reactions, surfactants slowed down ("inhibited") the reactions relative to the rate in the absence of surfactant, despite effective emulsification of the substrate. Surfactants varied in the extent to which the reaction was inhibited and inhibition occurred below the critical micelle concentration of surfactants. Inhibition was accompanied by a loss of enantioselectivity with the crude enzyme but not the purified enzyme, indicating the presence of more than one activity in the crude PPL preparation. In general, there would seem to be no advantage to be gained from the use of surfactants in the hydrolysis of compounds of low water solubility with lipolytic enzymes; the use of an immiscible cosolvent is more effective. The pyruvate decarboxylase (EC 4.1.1.1) from Zymomonas mobilis strain CP4 ATCC 31821 was purified from recombinant Escherichia coli harbouring the plasmid pLOI295, which contained the gene coding for the enzyme. The purified recombinant enzyme catalysed acyloin condensations with a number of aldehyde acceptors. The substrate specificity of the Zymomonas enzyme was very similar to that observed with the enzyme from Saccharomyces carlsbergensis. However, the Zymomonas enzyme was found to catalyse the formation of acyloins from acetaldehyde at a rate four orders of magnitute greater than that observed with yeast enzyme. By comparing the stereochemistry of acyloin condensations catalysed by the Zymomonas and yeast enzymes, differences in the architecture of the active sites of these closely related enzymes have emerged.

R-phenylacetylcarbinol (PAC) is used as a precursor for production of ephedrine and pseudoephedrine, which are anti-asthmatics and nasal decongestants. PAC is produced from benzaldehyde and pyruvate mediated by pyruvate decarboxylase (PDC). A strain of Rhizopus javanicus was evaluated for its production of PDC. The morphology of R. javanicus was influenced by the degree of aeration/agitation. A relatively high specific PDC activity (328 U decarboxylase g-1 mycelium) was achieved when aeration/agitation were reduced significantly in the latter stages of cultivation. The stability of partially purified PDC and crude extract from R. javanicus were evaluated by examining the enzyme deactivation kinetic in various conditions. R. javanicus PDC was less stable than Candida utilis PDC currently used in our group. A kinetic model for the deactivation of partially purified PDC extracted from C. utilis by benzaldehyde (0?00 mM) in 2.5 M MOPS buffer has been developed. An initial lag period prior to deactivation was found to occur, with first order dependencies of PDC deactivation on exposure time and on benzaldehyde concentration. A mathematical model for the enzymatic biotransformation of PAC and its associated by-products has been developed using a schematic method devised by King and Altman (1956) for deriving the rate equations. The rate equations for substrates, product and by-products have been derived from the patterns for yeast PDC and combined with a deactivation model for PDC from C. utilis. Initial rate and biotransformation studies were applied to refine and validate a mathematical model for PAC production. The rate of PAC formation was directly proportional to the enzyme activity level up to 5.0 U carboligase ml-1. Michaelis-Menten kinetics were determined for the effect of pyruvate concentration on the reaction rate. The effect of benzaldehyde on the rate of PAC production followed the sigmoidal shape of the Monod-Wyman-Changeux (MWC) model. The biotransformation model, which also included a term for PDC inactivation by benzaldehyde, was used to determine the overall rate constants for the formation of PAC, acetaldehyde and acetoin. Implementation of digital pH control for PAC production in a well-stirred organic-aqueous two-phase biotransformation system with 20 mM MOPS and 2.5 M dipropylene glycol (DPG) in aqueous phase resulted in similar level of PAC production [1.01 M (151 g l-1) in an organic phase and 115 mM (17.2 g l-1) in an aqueous phase after 47 h] to the system with a more expensive 2.5 M MOPS buffer.

Rhizopus oryzae is a filamentous fungi which produces lactic acid and ethanol in fermentations. R. oryzae has numerous advantages for use industrial production of L-(+)-lactic acid but the yield of lactic acid produced on the basis of carbon consumed is low. Metabolic flux analysis of R. oryzae has shown that most of the pyruvate produced at the end of the glycolysis is channelled to ethanol, acetyl-CoA and oxaloacetate production. This study aimed to answer some questions addressed on the regulation of pyruvate branch point in R. oryzae and for this purpose biochemical characterisation of the enzymes acting at this branch point and cloning the genes coding for these enzymes have been done. Pyruvate decarboxylase was purified and characterised for the first time from R. oryzae. The purified enzyme has a Hill coefficient of 1.84 and the Km of the enzyme is 8.6 mM for pyruvate at pH 6.5. The enzyme is inhibited at pyruvate concentrations higher than 30 mM. The optimum pH for enzyme activity shows a broad range from 5.7 and 7.2. The monomer molecular weight was estimated as 59±
2 kDa by SDS-PAGE analysis. Pyruvate decarboxylase (pdcA and pdcB) and lactate dehydrogenase (ldhA and ldhB) genes of R. oryzae have been cloned by PCR-cloning approach and the filamentous fungi Aspergillus niger was transformed with these genes. The A. niger transformed with either of the ldh genes of R. oryzae showed enhanced production of lactic acid compared to wild type. Citric acid production was also increased in these transformants while no gluconate production was observed Cloning of hexokinase gene from R. oryzae using degenerate primers was studied by the use of GenomeWalker kit (Clontech). The results of this study were evaluated by using some bioinformatics tools depending on the unassembled clone sequences of R. oryzae genome.

Decarboxylation is an essential process in catabolic metabolism of essentially all nutrients that serve as sources of energy in biological cells and organisms. The most widely known biological process leading to decarboxylation is the metabolism of glucose, in which all of the carbon in the molecule is oxidized to carbon dioxide by way of the glycolytic pathway, the pyruvate dehydrogenase complex, and the tricarboxylic acid cycle. The decarboxylation steps take place in thiamine pyrophosphate (TPP)–dependent α-ketoacid dehydrogenase complexes and isocitrate dehydrogenase. The latter enzyme does not require a coenzyme, other than the cosubstrate NAD+. Many other decarboxylations require coenzymes such as pyridoxal-5'-phosphate (PLP) or a pyruvoyl moiety in the peptide chain. Biological carboxylation is the essential process in the fixation of carbon dioxide by plants and of bicarbonate by animals, plants, and bacteria. Carboxylation by enzymes requires the action of biotin or a divalent metal cofactor, and it requires ATP when the carboxylating agent is the bicarbonate ion. The most prevalent enzymatic carboxylation is that of ribulose bisphosphate carboxylase (rubisco), which is responsible for carbon dioxide fixation in plants. The basic chemistry of decarboxylation is illustrated by mechanisms A to D in fig. 8-1. The mechanisms all require some means of accommodation for the electrons from the cleavage of the bond linking the carboxylate group to the α-carbon. In mechanism A, an electron sink at the β-carbon provides a haven for two electrons. Acetoacetate decarboxylase functions by this mechanism (see chap. 1), as well as PLP- and TPP-dependent decarboxylases (see chap. 3). In mechanism B, a leaving group at the β-carbon departs with two electrons. Mevalonate-5-diphosphate decarboxylate functions by mechanism B and is discussed in a later section. In mechanism C, a leaving group replaces the α-carbon and departs with a pair of electrons. A biological example is formate dehydrogenase, in which the leaving group is a hydride that is transferred to NAD+. In mechanism D, a free radical center is created adjacent to the α-carbon and potentiates the homolytic scission of the bond to the carboxylate group. Mechanism D requires secondary electron transfer processes to create the radical center and quench the formyl radical.

The timing of dormancy induction and release is very important to the economic production of table grape. Advances in manipulation of dormancy induction and dormancy release are dependent on the establishment of a comprehensive understanding of biological mechanisms involved in bud dormancy. To gain insight into these mechanisms we initiated the research that had two main objectives: A. Analyzing the expression profiles of large subsets of genes, following controlled dormancy induction and dormancy release, and assessing the role of known metabolic pathways, known regulatory genes and novel sequences involved in these processes B. Comparing expression profiles following the perception of various artificial as well as natural signals known to induce dormancy release, and searching for gene showing similar expression patterns, as candidates for further study of pathways having potential to play a central role in dormancy release. We first created targeted EST collections from V. vinifera and V. riparia mature buds. Clones were randomly selected from cDNA libraries prepared following controlled dormancy release and controlled dormancy induction and from respective controls. The entire collection (7920 vinifera and 1194 riparia clones) was sequenced and subjected to bioinformatics analysis, including clustering, annotations and GO classifications. PCR products from the entire collection were used for printing of cDNA microarrays. Bud tissue in general, and the dormant bud in particular, are under-represented within the grape EST database. Accordingly, 59% of the our vinifera EST collection, composed of 5516 unigenes, are not included within the current Vitis TIGR collection and about 22% of these transcripts bear no resemblance to any known plant transcript, corroborating the current need for our targeted EST collection and the bud specific cDNA array. Analysis of the V. riparia sequences yielded 814 unigenes, of which 140 are unique (keilin et al., manuscript, Appendix B). Results from computational expression profiling of the vinifera collection suggest that oxidative stress, calcium signaling, intracellular vesicle trafficking and anaerobic mode of carbohydrate metabolism play a role in the regulation and execution of grape-bud dormancy release. A comprehensive analysis confirmed the induction of transcription from several calcium–signaling related genes following HC treatment, and detected an inhibiting effect of calcium channel blocker and calcium chelator on HC-induced and chilling-induced bud break. It also detected the existence of HC-induced and calcium dependent protein phosphorylation activity. These data suggest, for the first time, that calcium signaling is involved in the mechanism of dormancy release (Pang et al., in preparation). We compared the effects of heat shock (HS) to those detected in buds following HC application and found that HS lead to earlier and higher bud break. We also demonstrated similar temporary reduction in catalase expression and temporary induction of ascorbate peroxidase, glutathione reductase, thioredoxin and glutathione S transferase expression following both treatments. These findings further support the assumption that temporary oxidative stress is part of the mechanism leading to bud break. The temporary induction of sucrose syntase, pyruvate decarboxylase and alcohol dehydrogenase indicate that temporary respiratory stress is developed and suggest that mitochondrial function may be of central importance for that mechanism. These finding, suggesting triggering of identical mechanisms by HS and HC, justified the comparison of expression profiles of HC and HS treated buds, as a tool for the identification of pathways with a central role in dormancy release (Halaly et al., in preparation). RNA samples from buds treated with HS, HC and water were hybridized with the cDNA arrays in an interconnected loop design. Differentially expressed genes from the were selected using R-language package from Bioconductor project called LIMMA and clones showing a significant change following both HS and HC treatments, compared to control, were selected for further analysis. A total of 1541 clones show significant induction, of which 37% have no hit or unknown function and the rest represent 661 genes with identified function. Similarly, out of 1452 clones showing significant reduction, only 53% of the clones have identified function and they represent 573 genes. The 661 induced genes are involved in 445 different molecular functions. About 90% of those functions were classified to 20 categories based on careful survey of the literature. Among other things, it appears that carbohydrate metabolism and mitochondrial function may be of central importance in the mechanism of dormancy release and studies in this direction are ongoing. Analysis of the reduced function is ongoing (Appendix A). A second set of hybridizations was carried out with RNA samples from buds exposed to short photoperiod, leading to induction of bud dormancy, and long photoperiod treatment, as control. Analysis indicated that 42 genes were significant difference between LD and SD and 11 of these were unique.