Tesi sul tema "Pyruvate decarboxylase"

(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.

Um gene que codifica uma piruvato/indolpiruvato descarboxilase (PDC/IPDC) está presente no tripanossomatídeo de plantas Phytomonas serpens. A PDC atua na fermentação alcoólica, enquanto que a IPDC atua na biossíntese do fitormônio ácido indol-3-acético (AIA). Análises filogenéticas indicam que a PDC/IPDC de P. serpens é monofilética com IPDCs de gama-proteobactérias, sugerindo um evento de transferência horizontal gênica. A análise de meios de cultura de P. serpens confirma a produção de etanol e AIA. A funcionalidade do fitormônio foi confirmada em ensaios de alongamento de hipocótilos de tomateiros. Tomates inoculados com P. serpens mostraram aumento no teor de AIA-amida e -éster conjugados. A atividade PDC foi mostrada em extratos de P. serpens. Concluímos que a PDC/IPDC seria uma 2-cetoácido descaboxilase com atividade catalítica variável para diferentes substratos. A atividade PDC parece ser predominante em P. serpens, representando um mecanismo para oxidar parte do NADH formado na glicólise, principal responsável pela produção de ATP neste organismo.
A gene codifying a pyruvate/indolepyruvate decarboxylase (PDC/IPDC) is present in the plant trypanosomatid Phytomonas serpens. PDC acts in the alcoholic fermentation, whyle IPDC acts in the biosynthesis of the phytohormone indole-3-acetic acid (IAA). Phylogenetic analysis indicate that P. serpens PDC/IPDC is monophyletic with gamma-proteobacteria IPDCs, suggesting a horizontal gene transfer event. Analysis of P. serpens culture media confirms production of ethanol and IAA. The functionality of the phytohormone was confirmed by tomato hypocotyl elongation tests. Tomatoes inoculated with P. serpens showed an increase in the concentration of IAA amide and ester conjugated. PDC activity was shown in P. serpens extracts. We conclude that the PDC/IPDC would be a 2-keto acid decaboxylase with variable catalytic activity for different substrates. The PDC activity appears to be prevalent in P. serpens representing a mechanism to oxidize part of NADH formed in glycolysis, responsible for ATP production in this organism.

Extreme thermophiles and hyperthermophiles are microorganisms capable of growing optimally at 65-79??C and 80??C plus, respectively. Many of the enzymes isolated from them are thermostable, which makes them a potential resource for research and industrial applications. An increasing number of hyper/thermophiles is shown to be able to produce ethanol as an end-metabolite. Despite characterization of many alcohol dehydrogenases (ADHs) with a potential role in the production of ethanol, to date there has been no significant progress in identifying the enzymes responsible for the production of acetaldehyde, which is an intermediate in production of ethanol from pyruvate.
Pyruvate decarboxylase (PDC encoded by pdc) is a thiamine pyrophosphate (TPP)-containing enzyme responsible for conversion of pyruvate to acetaldehyde in many mesophilic organisms. However, no pdc/PDC homolog has yet been found in fully sequenced genomes of hyper/thermophiles. The only PDC activity reported in hyperthermophiles is a bifunctional, TPP- and CoA-dependent pyruvate ferredoxin oxidoreductase (POR)/PDC enzyme from the hyperthermophilic archaeon Pyrococcus furiosus.
The bifunctional and TPP-containing POR/PDC enzyme was isolated and characterized from the ethanol-producing hyperthermophilic archaeon Thermococcus guaymasensis (Topt=88??C), as well as the bacteria Thermotoga hypogea (Topt=70??C) and Thermotoga maritima (Topt=80??C). The T. guaymasensis enzyme was purified anaerobically to homogeneity as judged by SDS-PAGE analysis. POR and PDC activities were co-eluted from each of the chromatographic columns, and the ratio of POR to PDC activities remained constant throughout the purification steps. All of the enzyme activities were CoA- and TPP-dependent and highly sensitive toward exposure to air. The apparent kinetic parameters were determined for the main substrates, including pyruvate and CoA for each activity. Since the genome sequence of T. guaymasensis and T. hypogea were not available, sequences of the genes encoding POR were determined via primer walking and inverse PCR.
A novel enzyme capable of catalyzing the production of acetaldehyde from pyruvate in hyperthermophiles was also characterized. The enzyme contained TPP and flavin and was expressed as recombinant histidine-tagged protein in the mesophilic host Escherichia coli. The new enzyme was a bifunctional enzyme catalyzing another reaction as the major reaction besides catalyzing the non-oxidative decarboxylation of pyruvate to acetaldehyde.
Another enzyme known to be involved in catalysis of acetaldehyde production from pyruvate is CoA-acetylating acetaldehyde dehydrogenase (AcDH encoded by mhpF and adhE). Pyruvate is oxidized into acetyl-CoA by either POR or pyruvate formate lyase (PFL), and AcDH catalyzes the reduction of acetyl-CoA to acetaldehyde. AcDH is present in some mesophilic (such as clostridia) and thermophilic bacteria (e.g. Geobacillus and Thermoanaerobacter). However, no AcDH gene or protein homologs could be found in the released genomes of hyperthermophiles. Moreover, no such activity was detectable from the cell-free extracts of different hyperthermophiles used in this study.
In conclusion, no commonly-known PDCs was found in hyperthermophiles, but two types of acetaldehyde-producing enzymes were present in various bacterial and archaeal hyperthermophiles. Although the deduced amino acid sequences from different hyperthermophiles are quite similar, the levels of POR and PDC activities appeared to vary significantly between the archaeal and bacterial enzymes, which most likely reflects the different physiological implications of each activity.

Rice plants are partially or completely submerged when fields are flooded. During submergence, rice plants encounter anaerobic conditions, and suffer severe injury, often death, leading to major crop losses in countries affected by monsoonal flooding. Pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) catalyse thanol fermentation (EF), the major energy-producing pathway under conditions of low oxygen and are among the anaerobic polypeptides induced under such conditions. The importance of EF is emphasised by the reduced survival and germination of the ADH null mutants of maize, barley, rice and Arabidopsis under anaerobic conditions. The research described in this thesis has taken a transgenic approach to manipulate the levels of PDC and ADH to determine whether altering the EF pathway can affect anaerobic tolerance in rice. It was found that one antisense ADHl (rice gene)line had decreased levels of both ADHl and ADH2 polypeptides, and greatly reduced ADH activity of 6% of that of wild type (WT- untransformed Taipei). This antisense ADHI line showed reduced ethanol production and coleoptile growth under anoxia, and mature plants exhibited reduced survival when submerged in anaerobic water, suggesting ADH plays a role in seed germination and plant survival under anoxia. One sense ADH2 (cotton gene) line had significantly increased levels of ADH activity compared to WT and a flooding tolerant rice variety FR13A in air and under hypoxia. No significant increase in ethanol production was observed in the line which overproduced ADH by 439% of WT. Similar levels of anoxia tolerance were found in mature plants of the line which over-produced ADH and WT whereas in the flooding tolerant variety, anoxia tolerance was much higher. This suggests that over-production of ADH increases neither ethanol production nor anaerobic survival. Three independently transformed lines of the rice PDCl driven by an anaerobically inducible promoter contained an increased level of PDC1 polypeptides. A moderate increase in PDC activity and ethanol production compared to WT was also observed in these lines under anaerobic conditions. Effects of anoxia on seed germination were assayed in these lines over-producing PDC and neither retardation nor acceleration of germination was observed. However, mature plants showed decreased survival under anaerobiosis. On the contrary, hybrid plants over-expressing both PDC and ADH were found to have better anaerobic tolerance than plants over-producing PDC alone. These results indicate that overproducing PDC plants suffered from some kind of toxicity which was counterbalanced and/or neutralised in plants over-producing ADH along with PDC. Acetaldehyde levels were appreciably higher in the plants over-producing PDC compared to WT and hybrid plants over-producing both PDC and ADH indicating that acetaldehyde might cause early senescence in plants over-producing PDC alone under anaerobic conditions. No transformed lines with either over-producing PDC, ADH or both PDC and ADH had increased submergence tolerance relative to the WT, however lines often had different metabolic rates and demonstrated the versatility of a molecular approach to evaluating metabolic controls affecting plant growth and survival. A second objective of this research was to study the expression of the rice PDCl and PDC3 promoters in various tissues of rice by GUS histochemical analysis. Translational fusion of the PDCl promoter-GUS gave positive blue staining in embryos, endosperm, shoots, and roots and showed strong anaerobic induction in shoots and roots. GUS staining was found in anthers but absent in pollen. In immunoblotting analysis using an antibody raised against the rice PDC1 polypeptide, the PDC band corresponding to PDCl was also absent in pollen of untransformed Taipei, suggesting that the rice PDCl gene is not expressed in pollen. Nine independently transformed lines of the rice PDC3 promoter-GUS fusion (translational) did not express GUS in any vegetative tissues even under anaerobic conditions. GUS staining was seen in the pollen of three independently transformed lines of PDC3-GUS. A novel PDC band with a MWt of approx. 62 kDa was found in immunoblots of pollen of untransformed Taipei using an antibody generated against the rice PDCl, indicating that the rice PDC3 has pollen specific expression. Expression of PDC3 was seen after the first mitosis of microspores and increased with maturation, implying that it may have a role during pollen germination. The final objective was to study the cis-acting regulatory elements required for anaerobic induction in the rice PDCl promoter. GUS histochemical analysis of transcriptional fusions of the various lengths of 5' truncated PDCl promoter-GUS revealed that the regulation of the GUS reporter gene did not mirror that of the endogenous PDCl and the PDCl promoter acted in a constitutive manner.

Indiana University-Purdue University Indianapolis (IUPUI)
Thiamin diphosphate (ThDP)-dependent enzymes catalyze a wide range of reactions including the oxidative and nonoxidative decarboxylation of 2-keto acids, carboligation reactions, the cleavage of C-C bonds, and the formation of C-S, C-N, and C-O bonds. Surprisingly, given this diversity, all ThDP-dependent enzyme catalyzed reactions proceed through essentially the same intermediate. This suggests that these enzymes share a common ancestry and have evolved to become the diverse group of enzymes seen today. Sequence alignments have revealed that all ThDP-dependent enzymes share two common ThDP binding domains, the PYR domain and the PP domain. In addition to these conserved domains, over time, other domains have been added creating further diversity in this superfamily. For instance, the TH3 domain, found in many ThDP-dependent enzymes, serves the function of binding additional cofactors such as FAD in enzymes like acetohydroxyacid synthase (AHAS) but in others, like pyruvate decarboxylase (PDC), it has lost this function completely. The work presented here focuses on ThDP-dependent decarboxylases. In this thesis, several evolutionary aspects of this group of enzymes will be examined including (i) the characterization of an evolutionary forerunner in the presence of a mechanism-based inhibitor, (ii) the characterization of the minor isozymes of pyruvate decarboxylase from Saccharomyces cerevisiae, and (iii) the development of a selection method to increase the efficiency of the site-saturation mutagenesis used to study ThDP-dependent enzyme evolution.

Methanogens, members of the domain Archaea, are unique in their ability to reduce carbon substrates to methane. Coenzyme M (CoM) is required in all methanogenic pathways. The biosynthesis of this coenzyme has been well studied in Class I Methanogens, but in Class II Methanogens, such as Methanosarcina acetivorans, little is known. The first step in the biosynthetic pathway might be catalyzed by cysteate synthase (CS), which converts phosphoserine to cysteate by the addition of sulfite. The 46 kDa enzyme was successfully purified from inclusion bodies and characterized. The identity of the product was confirmed by liquid chromatography-mass spectrometry (LC-MS) results as well as by derivatization of the reaction product coupled with high pressure liquid chromatography (HPLC) analysis. Kinetic analysis showed that the enzyme has a K [subscript m] of 0.43 mM for its substrate, phosphoserine, and a K [subscript m] of 0.05 mM for its required nucleophile, sulfite. Four compounds were found to be inhibitors and IC₅₀ values were determined. The results show that CS carries out a new reaction and narrows the gap in our knowledge of Class II Methanogen CoM biosynthesis. In the second part of this dissertation, five enzymes in a newly discovered but poorly characterized pathway for the degradation of halogenated aromatic compounds in Leptothrix cholodnii SP-6 were examined. The pathway reportedly culminates in the production of 2-chloroacetaldehyde, a well-known alkylating agent. In order to determine if 2-chloroacetaldehyde is produced and how the organism survives in its presence, the pathway intermediates are being identified. To this end, 4-oxalocrotonate tautomerase (4-OT), 4-oxalocrotonate decarboxylase (4-OD), vinylpyruvate hydratase (VPH), pyruvate aldolase (PA) and acetaldehyde dehydrogenase (AAD) were cloned, expressed and characterized. 4-OT was found to process the 5-(chloro)-2-hydroxymuconate, but only when the equilibrium was shifted by the addition of 4-OD and VPH. Steady state kinetic analysis showed that while there is a slight decrease in K [subscript m] for the halogenated substrate when compared to the non-halogenated substrate, indicating a difference in binding. There is also a 30-fold decrease in the turnover number, indicating a preference for the non-halogenated substrate. The identity of the product, 5-(chloro)-2-oxo-4-hydroxypentanoate, was verified by ¹H NMR spectroscopy. A stereochemical analysis was also carried out.
text

The evolution of alcoholic fermentation in the non-conventional Wickerhamiella/Starmerella (W/S) yeast clade is characterized by the loss of native pyruvate decarboxylase (PDC1) and alcohol dehydrogenase (ADH) genes. In some species, the reacquisition of this via was achieved through horizontal gene transfer (HGT) of ADH and co-option of a native decarboxylase, Aro10. This work aimed to build on the previous knowledge regarding alcoholic fermentation in the W/S clade, by combining in silico data obtained from newly sequenced genomes, with phenotypic assays, aiming to assess alcoholic fermentation abilities and characterization of Adh enzymes, in a subset of species. Three independent HGT events were previously reported to have introduced different bacterial ADH1 in distinct subgroups of the W/S clade. Subgroups A, B and C harbour ADH1a, ADH1b and ADH1c, respectively. Subgroup ADH0 does not carry an ADH1. In this work, data supporting these three HGT events was obtained and two new HGT events of bacterial ADH6 were detected. Most ADH6 genes were acquired in the ancestor of the subgroups reported for ADH1, while one was found in a ADH0 species (Wickerhamiella slavikovae) that seemingly lacks other alcoholic fermentation genes (ADH1, PDC1 and ARO10). As for the remaining species, while ARO10 is present, PDC1 is absent. Ethanol production was generally observed in subgroup A, while its assimilation was verified in subgroups B and C, suggesting that Adh proteins are functional. It was confirmed that Adh1a from Starmerella bombicola is involved in the interconversion of acetaldehyde and ethanol, using NAD(H) and NADP(H) as cofactors, which contrasts with the specificity of Adh proteins from yeasts towards NAD(H). To further understand the evolution of the alcoholic fermentation in the W/S clade, it is essential to combine comparative genomics with the characterization of these enzymes to evaluate their role on the central carbon metabolism.
A evolução da fermentação alcoólica no clado de leveduras não convencionais Wickerhamiella/Starmerella (W/S) é caracterizada pela perda dos genes nativos da piruvato descarboxilase (PDC1) e das álcool desidrogenases (ADH). Em algumas espécies, a reaquisição desta via foi conseguida através de transferências horizontais de genes (HGT) ADH e pela cooptação de uma descarboxilase nativa, Aro10. Este trabalho teve como objetivo partir do conhecimento prévio relativo à fermentação alcoólica no clado W/S, combinando dados in silico de novos genomas sequenciados, com ensaios fenotípicos, de forma a avaliar as capacidades fermentativas e caracterizar as enzimas Adh, num conjunto de espécies. Três eventos HGT independentes foram previamente identificados como tendo introduzido diferentes ADH1 bacterianos nos distintos subgrupos do clado W/S. Os subgrupos A, B e C possuem ADH1a, ADH1b e ADH1c, respetivamente. O subgrupo ADH0 não possui ADH1. Neste trabalho, dados que suportam estes três eventos HGT foram obtidos e dois novos eventos HGT de ADH6 bacterianos foram detetados. A maioria dos genes ADH6 foram adquiridos nos mesmos ancestrais dos subgrupos reportados para ADH1, enquanto um foi encontrado numa espécie ADH0 (Wickerhamiella slavikovae) que aparentemente não possui outros genes da fermentação alcoólica (ADH1, PDC1 e ARO10). Em relação às espécies restantes, enquanto ARO10 está presente, PDC1 está ausente. A produção de etanol foi geralmente observada no subgrupo A, enquanto a sua assimilação foi verificada nos subgrupos B e C, sugerindo que as proteínas Adh são funcionais. Foi confirmado o papel da Adh1a de Starmerella bombicola na interconversão de acetaldeído e etanol, usando NAD(H) e NADP(H) como cofatores, o que contrasta com a especificidade das proteínas Adh de leveduras relativamente a NAD(H). Para compreender melhor a evolução da fermentação alcoólica no clado W/S, é essencial combinar genómica comparativa com a caracterização destas enzimas, de forma a avaliar o seu papel no metabolismo central de carbono.

The tautomerase superfamily is composed of a group of proteins characterized by two key features: the N-terminal proline and a beta-alpha-beta-motif. This superfamily has been divided into five families represented by 4-oxalocrotonate tautomerase (4-OT), 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), cis-3-chloroacrylic acid dehalogenase (cis-CaaD), malonate semialdehyde decarboxylase (MSAD), and macrophage migration inhibitory factor (MIF). Cg10062 is a homologue of cis-CaaD, but has several distinct biochemical properties from cis-CaaD. For example, Cg10062 can be irreversibly inhibited by (R)- or (S)-oxirane-2-carboxylate, whereas cis-CaaD can only be irreversibly inhibited by (R)-oxirane-2-carboxylate. FG41MSAD is a homologue of MSAD, with comparable decarboxylase activity but missing Arg-73 known to be crucial for the MSAD activity. In order to understand the unique biochemical characteristics of Cg10062 and FG41MSAD, we have solved five crystal structures. These crystal structures have established a solid structural basis for understanding the mechanisms of their activities. The eukaryotic protein phosphatases are composed of a group of proteins that are responsible for reversible phosphorylation. The eukaryotic protein phosphatases have been divided into three families, the phosphoprotein phosphatase (PPP) family, the protein phosphatase Mg2+- or Mn2+-dependent (PPM) family and the protein Tyr phosphatase (PTP) family. PDP1 is a member of PPM family. PDP1 is also an important component of the large pyruvate dehydrogenase complex (PDC) which catalyzes the decarboxylation of pyruvate to yield acetyl-CoA with the accompanying reduction of NAD+. In order to understand the mechanism in which it dephosphorylates its target protein we have solved the structure of the catalytic domain of PDP1. Analysis of these structures in the light of their evolutionary contexts enables us to appreciate the unity and diversity of the biological systems at the chemical level and help us solve interesting problems, such as the possible physiological functions for some members within the tautomerase superfamily.
text