No description available.

(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.

The synthesis of a new thiamine analogue, 2'-hydroxythiamine, is reported. Kinetic studies with thiamine pyrophosphate analogues and apopyruvate decarboxylase (EC 4.1.1.1) from brewer's yeast, gave the values of 2.3 X 10^-5 M as the K_m for thiamine pyrophosphate and 2.0 X 10^-5 M as the K_m for 2'-ethylthiamine pyrophosphate. The V_max for the latter was 14% that of thiamine pyrophosphate. Inhibitor constants, K_i, were determined for the following competitive inhibitors of thiamine pyrophosphate with the apoenzyme. All values are given for the pyrophosphate esters: tetrahydrothiamine, 0.65 X 10^-5 M; oxythiamine, 2.0 X 10^-5 M; 2'-n-butylthiamine, 4.5 X 10^-5 M; 2'-methoxythiamine, 7.0 X 10^-5 M; pyrithiamine, 7.8 X 10^-5; thiochrome, 15. X 10^-5 M; 2'-desmethylthiamine, 22. X 10^-5 M; 2'-hydroxythiamine, 38. X 10^-5 M. None of the inhibitors exhibited coenzyme activity. A hydrophobic interaction of the 2'-methyl group of thiamine pyrophosphate with the apoenzyme is suggested from these studies. The formation of a fluorescent complex at pH 6.7 between apopyruvate decarboxylase and thiochrome pyrophosphate was detected and found to be dependent upon Mg(II) ions. A similar complex between thiochrome and the apoenzyme could not be detected, demonstrating the importance of the pyrophosphate function in binding to the protein. The shift in the fluorescence emission spectrum of thiochrome pyrophosphate toward lower wavelengths upon complex formation with the apoenzyme, coincided with the behavior of thiochrome in solvents of decreasing dielectric constant. This latter observation suggests involvement of the thiochrome pyrophosphate with a hydrophobic region of the enzyme. A study of the pH dependency of the enzyme-coenzyme complex indicated considerable recombination of apoenzyme and coenzyme at alkaline pH, where dissociation of the coenzyme usually takes place. A rationale for the interpretation of the pH-behavior of the enzyme-coenzyme complex is offered. An amino acid analysis of a highly purified sample of pyruvate decarboxylase, considered to be essentially homogeneous, is reported. Assuming a molecular weight of 175,000 for the enzyme, a total of 1317 amino acid residues were calculated, of which 52.1% fall into the non-polar category. the half-cystine content was calculated as 10.3%, and the proline content, as 4.6%. The specific volume was calculated as 0.737 ml per g. A single low-angle X-ray diffraction study gave a value of 35.5 ± 1.5 A for the radius of gyration of pyruvate decarboxylase. Assuming a spherical shape, a diameter of 91.6 ± 4.0 A was calculated.

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.

No description available.

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.<br> 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.<br> 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.<br> 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.<br> 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.<br> 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.

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.

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.

No description available.

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.