The Experts below are selected from a list of 291 Experts worldwide ranked by ideXlab platform
Pere Clapés - One of the best experts on this subject based on the ideXlab platform.
-
structure guided redesign of d fructose 6 phosphate aldolase from e coli remarkable activity and selectivity towards acceptor substrates by two point mutation
Chemical Communications, 2011Co-Authors: Mariana L Gutierrez, Jordi Bujons, Pere ClapésAbstract:Structure-guided re-design of the acceptor binding site of D-fructose-6-phosphate aldolase from E. coli leads to the construction of FSA A129S/A165G double mutant with an activity between 5- to >900-fold higher than that of wild-type towards N-Cbz-Aminoaldehyde derivatives.
-
dihydroxyacetone phosphate aldolase catalyzed synthesis of structurally diverse polyhydroxylated pyrrolidine derivatives and evaluation of their glycosidase inhibitory properties
Chemistry: A European Journal, 2009Co-Authors: Jordi Calveras, Jordi Bujons, Meritxell Egidogabas, Livia Gomez, Josefina Casas, Teodor Parella, Jesus Joglar, Pere ClapésAbstract:Abstract The chemoenzymatic synthesis of a collection of pyrrolidine-type iminosugars generated by the aldol addition of dihydroxyacetone phosphate (DHAP) to C-alpha-substituted N-Cbz-2-Aminoaldehydes derivatives, catalyzed by DHAP aldolases is reported. L-fuculose-1-phosphate aldolase (FucA) and L-rhamnulose-1-phosphate aldolase (RhuA) from E. coli were used as biocatalysts to generate configurational diversity on the iminosugars. Alkyl linear substitutions at C-alpha were well tolerated by FucA catalyst (i.e., 40-70 % conversions to aldol adduct), whereas no product was observed with C-alpha-alkyl branched substitutions, except for dimethyl and benzyl substitutions (20 %). RhuA was the most versatile biocatalyst: C-alpha-alkyl linear groups gave the highest conversions to aldol adducts (60-99 %), while the C-alpha-alkyl branched ones gave moderate to good conversions (50-80 %), with the exception of dimethyl and benzyl substituents (20 %). FucA was the most stereoselective biocatalyst (90-100 % anti (3R,4R) adduct). RhuA was highly stereoselective with (S)-N-Cbz-2-Aminoaldehydes (90-100 % syn (i.e., 3R,4S) adduct), whereas those with R configuration gave mixtures of anti/syn adducts. For iPr and iBu substituents, RhuA furnished the anti adduct (i.e., FucA stereochemistry) with high stereoselectivity. Molecular models of aldol products with iPr and iBu substituents and as complexes with the RhuA active site suggest that the anti adducts could be kinetically preferred, while the syn adducts would be the equilibrium products. The polyhydroxylated pyrrolidines generated were tested as inhibitors against seven glycosidases. Among them, good inhibitors of alpha-L-fucosidase (IC50=1-20 microM), moderate of alpha-L-rhamnosidase (IC50=7-150 microM), and weak of alpha-D-mannosidase (IC50=80-400 microM) were identified. The apparent inhibition constant values (Ki) were calculated for the most relevant inhibitors and computational docking studies were performed to understand both their binding capacity and the mode of interaction with the glycosidases.
-
stereoselective aldol additions catalyzed by dihydroxyacetone phosphate dependent aldolases in emulsion systems preparation and structural characterization of linear and cyclic iminopolyols from Aminoaldehydes
Chemistry: A European Journal, 2003Co-Authors: Laia Espelt, Jordi Bujons, Teodor Parella, Jesus Joglar, Conxita Solans, Antonio Delgado, Pere ClapésAbstract:13 pages, 2 figures, 6 schemes, 5 tables.-- PMID: 14562306 [PubMed].-- Printed version published Oct 17, 2003.-- Supporting information available at: http://www.wiley-vch.de/contents/jc_2111/2003/f4966_s.pdf
Paraskevi Tavladoraki - One of the best experts on this subject based on the ideXlab platform.
-
functional diversity inside the arabidopsis polyamine oxidase gene family
Journal of Experimental Botany, 2011Co-Authors: Paola Fincato, Rodolfo Federico, Riccardo Angelini, Kalliopi A Roubelakisangelakis, Panagiotis N Moschou, Raffaela Tavazza, Valentina Spedaletti, Paraskevi TavladorakiAbstract:Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine catabolism. All so far characterized PAOs from monocotyledonous plants, such as the apoplastic maize PAO, oxidize spermine (Spm) and spermidine (Spd) to produce 1,3-diaminopropane, H2O2, and an Aminoaldehyde, and are thus considered to be involved in a terminal catabolic pathway. Mammalian PAOs oxidize Spm or Spd (and/or their acetyl derivatives) differently from monocotyledonous PAOs, producing Spd or putrescine, respectively, in addition to H2O2 and an Aminoaldehyde, and are therefore involved in a polyamine back-conversion pathway. In Arabidopsis thaliana, five PAOs (AtPAO1‐ AtPAO5) are present with cytosolic or peroxisomal localization and three of them (the peroxisomal AtPAO2, AtPAO3, and AtPAO4) form a distinct PAO subfamily. Here, a comparative study of the catalytic properties of recombinant AtPAO1, AtPAO2, AtPAO3, and AtPAO4 is presented, which shows that all four enzymes strongly resemble their mammalian counterparts, being able to oxidize the common polyamines Spd and/or Spm through a polyamine backconversion pathway. The existence of this pathway in Arabidopsis plants is also evidenced in vivo. These enzymes are also able to oxidize the naturally occurring uncommon polyamines norspermine and thermospermine, the latter being involved in important plant developmental processes. Furthermore, data herein reveal some important differences in substrate specificity among the various AtPAOs, which suggest functional diversity inside the AtPAO gene family. These results represent a new starting point for further understanding of the physiological role(s) of the polyamine catabolic pathways in plants.
-
inducible expression of maize polyamine oxidase in the nucleus of mcf 7 human breast cancer cells confers sensitivity to etoposide
Amino Acids, 2008Co-Authors: Lucia Marcocci, Pasquale Stano, Rodolfo Federico, M Casadei, Carmen Faso, A Antoccia, S Leone, B Mondovi, Paraskevi TavladorakiAbstract:In this study, polyamine oxidase from maize (MPAO), which is involved in the terminal catabolism of spermidine and spermine to produce an Aminoaldehyde, 1,3-diaminopropane and H2O2, has been conditionally expressed at high levels in the nucleus of MCF-7 human breast cancer cells, with the aim to interfere with polyamine homeostasis and cell proliferation. Recombinant MPAO expression induced accumulation of a high amount of 1,3-diaminopropane, an increase of putrescine levels and no alteration in the cellular content of spermine and spermidine. Furthermore, recombinant MPAO expression did not interfere with cell growth of MCF-7 cells under normal conditions but it did confer higher growth sensitivity to etoposide, a DNA topoisomerase II inhibitor widely used as antineoplastic drug. These data suggest polyamine oxidases as a potential tool to improve the efficiency of antiproliferative agents despite the difficulty to interfere with cellular homeostasis of spermine and spermidine.
-
heterologous expression and biochemical characterization of a polyamine oxidase from arabidopsis involved in polyamine back conversion
Plant Physiology, 2006Co-Authors: Paraskevi Tavladoraki, Fabio Polticelli, Riccardo Angelini, Marianna Nicoletta Rossi, Giuseppe Saccuti, Miguel A Perezamador, Rodolfo FedericoAbstract:Polyamine oxidase (PAO) is a flavin adenine dinucleotide-dependent enzyme involved in polyamine catabolism. Animal PAOs oxidize spermine (Spm), spermidine (Spd), and/or their acetyl derivatives to produce H2O2, an Aminoaldehyde, and Spd or putrescine, respectively, thus being involved in a polyamine back-conversion pathway. On the contrary, plant PAOs that have been characterized to date oxidize Spm and Spd to produce 1,3-diaminopropane, H2O2, and an Aminoaldehyde and are therefore involved in the terminal catabolism of polyamines. A database search within the Arabidopsis (Arabidopsis thaliana) genome sequence showed the presence of a gene (AtPAO1) encoding for a putative PAO with 45% amino acid sequence identity with maize (Zea mays) PAO. The AtPAO1 cDNA was isolated and cloned in a vector for heterologous expression in Escherichia coli. The recombinant protein was purified by affinity chromatography on guazatine-Sepharose 4B and was shown to be a flavoprotein able to oxidize Spm, norspermine, and N1-acetylspermine with a pH optimum at 8.0. Analysis of the reaction products showed that AtPAO1 produces Spd from Spm and norspermidine from norspermine, demonstrating a substrate oxidation mode similar to that of animal PAOs. To our knowledge, AtPAO1 is the first plant PAO reported to be involved in a polyamine back-conversion pathway.
-
functions of amine oxidases in plant development and defence
Trends in Plant Science, 2006Co-Authors: Alessandra Cona, Riccardo Angelini, Rodolfo Federico, Giuseppina Rea, Paraskevi TavladorakiAbstract:Copper amine oxidases and flavin-containing amine oxidases catalyse the oxidative de-amination of polyamines, which are ubiquitous compounds essential for cell growth and proliferation. Far from being only a means of degrading cellular polyamines and, thus, contributing to polyamine homeostasis, amine oxidases participate in important physiological processes through their reaction products. In plants, the production of hydrogen peroxide (H 2 O 2 ) deriving from polyamine oxidation has been correlated with cell wall maturation and lignification during development as well as with wound-healing and cell wall reinforcement during pathogen invasion. As a signal molecule, H 2 O 2 derived from polyamine oxidation mediates cell death, the hypersensitive response and the expression of defence genes. Furthermore, Aminoaldehydes and 1,3-diaminopropane from polyamine oxidation are involved in secondary metabolite synthesis and abiotic stress tolerance.
Rodolfo Federico - One of the best experts on this subject based on the ideXlab platform.
-
functional diversity inside the arabidopsis polyamine oxidase gene family
Journal of Experimental Botany, 2011Co-Authors: Paola Fincato, Rodolfo Federico, Riccardo Angelini, Kalliopi A Roubelakisangelakis, Panagiotis N Moschou, Raffaela Tavazza, Valentina Spedaletti, Paraskevi TavladorakiAbstract:Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine catabolism. All so far characterized PAOs from monocotyledonous plants, such as the apoplastic maize PAO, oxidize spermine (Spm) and spermidine (Spd) to produce 1,3-diaminopropane, H2O2, and an Aminoaldehyde, and are thus considered to be involved in a terminal catabolic pathway. Mammalian PAOs oxidize Spm or Spd (and/or their acetyl derivatives) differently from monocotyledonous PAOs, producing Spd or putrescine, respectively, in addition to H2O2 and an Aminoaldehyde, and are therefore involved in a polyamine back-conversion pathway. In Arabidopsis thaliana, five PAOs (AtPAO1‐ AtPAO5) are present with cytosolic or peroxisomal localization and three of them (the peroxisomal AtPAO2, AtPAO3, and AtPAO4) form a distinct PAO subfamily. Here, a comparative study of the catalytic properties of recombinant AtPAO1, AtPAO2, AtPAO3, and AtPAO4 is presented, which shows that all four enzymes strongly resemble their mammalian counterparts, being able to oxidize the common polyamines Spd and/or Spm through a polyamine backconversion pathway. The existence of this pathway in Arabidopsis plants is also evidenced in vivo. These enzymes are also able to oxidize the naturally occurring uncommon polyamines norspermine and thermospermine, the latter being involved in important plant developmental processes. Furthermore, data herein reveal some important differences in substrate specificity among the various AtPAOs, which suggest functional diversity inside the AtPAO gene family. These results represent a new starting point for further understanding of the physiological role(s) of the polyamine catabolic pathways in plants.
-
inducible expression of maize polyamine oxidase in the nucleus of mcf 7 human breast cancer cells confers sensitivity to etoposide
Amino Acids, 2008Co-Authors: Lucia Marcocci, Pasquale Stano, Rodolfo Federico, M Casadei, Carmen Faso, A Antoccia, S Leone, B Mondovi, Paraskevi TavladorakiAbstract:In this study, polyamine oxidase from maize (MPAO), which is involved in the terminal catabolism of spermidine and spermine to produce an Aminoaldehyde, 1,3-diaminopropane and H2O2, has been conditionally expressed at high levels in the nucleus of MCF-7 human breast cancer cells, with the aim to interfere with polyamine homeostasis and cell proliferation. Recombinant MPAO expression induced accumulation of a high amount of 1,3-diaminopropane, an increase of putrescine levels and no alteration in the cellular content of spermine and spermidine. Furthermore, recombinant MPAO expression did not interfere with cell growth of MCF-7 cells under normal conditions but it did confer higher growth sensitivity to etoposide, a DNA topoisomerase II inhibitor widely used as antineoplastic drug. These data suggest polyamine oxidases as a potential tool to improve the efficiency of antiproliferative agents despite the difficulty to interfere with cellular homeostasis of spermine and spermidine.
-
heterologous expression and biochemical characterization of a polyamine oxidase from arabidopsis involved in polyamine back conversion
Plant Physiology, 2006Co-Authors: Paraskevi Tavladoraki, Fabio Polticelli, Riccardo Angelini, Marianna Nicoletta Rossi, Giuseppe Saccuti, Miguel A Perezamador, Rodolfo FedericoAbstract:Polyamine oxidase (PAO) is a flavin adenine dinucleotide-dependent enzyme involved in polyamine catabolism. Animal PAOs oxidize spermine (Spm), spermidine (Spd), and/or their acetyl derivatives to produce H2O2, an Aminoaldehyde, and Spd or putrescine, respectively, thus being involved in a polyamine back-conversion pathway. On the contrary, plant PAOs that have been characterized to date oxidize Spm and Spd to produce 1,3-diaminopropane, H2O2, and an Aminoaldehyde and are therefore involved in the terminal catabolism of polyamines. A database search within the Arabidopsis (Arabidopsis thaliana) genome sequence showed the presence of a gene (AtPAO1) encoding for a putative PAO with 45% amino acid sequence identity with maize (Zea mays) PAO. The AtPAO1 cDNA was isolated and cloned in a vector for heterologous expression in Escherichia coli. The recombinant protein was purified by affinity chromatography on guazatine-Sepharose 4B and was shown to be a flavoprotein able to oxidize Spm, norspermine, and N1-acetylspermine with a pH optimum at 8.0. Analysis of the reaction products showed that AtPAO1 produces Spd from Spm and norspermidine from norspermine, demonstrating a substrate oxidation mode similar to that of animal PAOs. To our knowledge, AtPAO1 is the first plant PAO reported to be involved in a polyamine back-conversion pathway.
-
functions of amine oxidases in plant development and defence
Trends in Plant Science, 2006Co-Authors: Alessandra Cona, Riccardo Angelini, Rodolfo Federico, Giuseppina Rea, Paraskevi TavladorakiAbstract:Copper amine oxidases and flavin-containing amine oxidases catalyse the oxidative de-amination of polyamines, which are ubiquitous compounds essential for cell growth and proliferation. Far from being only a means of degrading cellular polyamines and, thus, contributing to polyamine homeostasis, amine oxidases participate in important physiological processes through their reaction products. In plants, the production of hydrogen peroxide (H 2 O 2 ) deriving from polyamine oxidation has been correlated with cell wall maturation and lignification during development as well as with wound-healing and cell wall reinforcement during pathogen invasion. As a signal molecule, H 2 O 2 derived from polyamine oxidation mediates cell death, the hypersensitive response and the expression of defence genes. Furthermore, Aminoaldehydes and 1,3-diaminopropane from polyamine oxidation are involved in secondary metabolite synthesis and abiotic stress tolerance.
Jordi Bujons - One of the best experts on this subject based on the ideXlab platform.
-
structure guided redesign of d fructose 6 phosphate aldolase from e coli remarkable activity and selectivity towards acceptor substrates by two point mutation
Chemical Communications, 2011Co-Authors: Mariana L Gutierrez, Jordi Bujons, Pere ClapésAbstract:Structure-guided re-design of the acceptor binding site of D-fructose-6-phosphate aldolase from E. coli leads to the construction of FSA A129S/A165G double mutant with an activity between 5- to >900-fold higher than that of wild-type towards N-Cbz-Aminoaldehyde derivatives.
-
dihydroxyacetone phosphate aldolase catalyzed synthesis of structurally diverse polyhydroxylated pyrrolidine derivatives and evaluation of their glycosidase inhibitory properties
Chemistry: A European Journal, 2009Co-Authors: Jordi Calveras, Jordi Bujons, Meritxell Egidogabas, Livia Gomez, Josefina Casas, Teodor Parella, Jesus Joglar, Pere ClapésAbstract:Abstract The chemoenzymatic synthesis of a collection of pyrrolidine-type iminosugars generated by the aldol addition of dihydroxyacetone phosphate (DHAP) to C-alpha-substituted N-Cbz-2-Aminoaldehydes derivatives, catalyzed by DHAP aldolases is reported. L-fuculose-1-phosphate aldolase (FucA) and L-rhamnulose-1-phosphate aldolase (RhuA) from E. coli were used as biocatalysts to generate configurational diversity on the iminosugars. Alkyl linear substitutions at C-alpha were well tolerated by FucA catalyst (i.e., 40-70 % conversions to aldol adduct), whereas no product was observed with C-alpha-alkyl branched substitutions, except for dimethyl and benzyl substitutions (20 %). RhuA was the most versatile biocatalyst: C-alpha-alkyl linear groups gave the highest conversions to aldol adducts (60-99 %), while the C-alpha-alkyl branched ones gave moderate to good conversions (50-80 %), with the exception of dimethyl and benzyl substituents (20 %). FucA was the most stereoselective biocatalyst (90-100 % anti (3R,4R) adduct). RhuA was highly stereoselective with (S)-N-Cbz-2-Aminoaldehydes (90-100 % syn (i.e., 3R,4S) adduct), whereas those with R configuration gave mixtures of anti/syn adducts. For iPr and iBu substituents, RhuA furnished the anti adduct (i.e., FucA stereochemistry) with high stereoselectivity. Molecular models of aldol products with iPr and iBu substituents and as complexes with the RhuA active site suggest that the anti adducts could be kinetically preferred, while the syn adducts would be the equilibrium products. The polyhydroxylated pyrrolidines generated were tested as inhibitors against seven glycosidases. Among them, good inhibitors of alpha-L-fucosidase (IC50=1-20 microM), moderate of alpha-L-rhamnosidase (IC50=7-150 microM), and weak of alpha-D-mannosidase (IC50=80-400 microM) were identified. The apparent inhibition constant values (Ki) were calculated for the most relevant inhibitors and computational docking studies were performed to understand both their binding capacity and the mode of interaction with the glycosidases.
-
stereoselective aldol additions catalyzed by dihydroxyacetone phosphate dependent aldolases in emulsion systems preparation and structural characterization of linear and cyclic iminopolyols from Aminoaldehydes
Chemistry: A European Journal, 2003Co-Authors: Laia Espelt, Jordi Bujons, Teodor Parella, Jesus Joglar, Conxita Solans, Antonio Delgado, Pere ClapésAbstract:13 pages, 2 figures, 6 schemes, 5 tables.-- PMID: 14562306 [PubMed].-- Printed version published Oct 17, 2003.-- Supporting information available at: http://www.wiley-vch.de/contents/jc_2111/2003/f4966_s.pdf
Marek Šebela - One of the best experts on this subject based on the ideXlab platform.
-
Plant ALDH10 family: identifying critical residues for substrate specificity and trapping a thiohemiacetal intermediate.
Journal of Biological Chemistry, 2013Co-Authors: David Kopecny, Radka Končitíková, Martina Tylichová, Armelle Vigouroux, Hana Moskalikova, Miroslav Soural, Marek Šebela, Solange MoréraAbstract:Abstract Plant ALDH10 family members are Aminoaldehyde dehydrogenases (AMADHs), which oxidize ω-Aminoaldehydes to the corresponding acids. They have been linked to polyamine catabolism, osmoprotection, secondary metabolism (fragrance), and carnitine biosynthesis. Plants commonly contain two AMADH isoenzymes. We previously studied the substrate specificity of two AMADH isoforms from peas (PsAMADHs). Here, two isoenzymes from tomato (Solanum lycopersicum), SlAMADHs, and three AMADHs from maize (Zea mays), ZmAMADHs, were kinetically investigated to obtain further clues to the catalytic mechanism and the substrate specificity. We also solved the high resolution crystal structures of SlAMADH1 and ZmAMADH1a because these enzymes stand out from the others regarding their activity. From the structural and kinetic analysis, we can state that five residues at positions 163, 288, 289, 444, and 454 (PsAMADHs numbering) can, directly or not, significantly modulate AMADH substrate specificity. In the SlAMADH1 structure, a PEG aldehyde derived from the precipitant forms a thiohemiacetal intermediate, never observed so far. Its absence in the SlAMADH1-E260A structure suggests that Glu-260 can activate the catalytic cysteine as a nucleophile. We show that the five AMADHs studied here are capable of oxidizing 3-dimethylsulfoniopropionaldehyde to the cryo- and osmoprotectant 3-dimethylsulfoniopropionate. For the first time, we also show that 3-acetamidopropionaldehyde, the third Aminoaldehyde besides 3-aminopropionaldehyde and 4-aminobutyraldehyde, is generally oxidized by AMADHs, meaning that these enzymes are unique in metabolizing and detoxifying aldehyde products of polyamine degradation to nontoxic amino acids. Finally, gene expression profiles in maize indicate that AMADHs might be important for controlling ω-Aminoaldehyde levels during early stages of the seed development.
-
carboxylate and aromatic active site residues are determinants of high affinity binding of ω Aminoaldehydes to plant Aminoaldehyde dehydrogenases
FEBS Journal, 2011Co-Authors: David Kopecny, Martina Tylichová, Jacques Snegaroff, Hana Popelkova, Marek ŠebelaAbstract:The crystal structures of both isoforms of the Aminoaldehyde dehydrogenase from pea (PsAMADH) have been solved recently [Tylichovaet al. (2010) J Mol Biol396, 870–882]. The characterization of the PsAMADH2 proteins, altered here by site-directed mutagenesis, suggests that the D110 and D113 residues at the entrance to the substrate channel are required for high-affinity binding of ω-Aminoaldehydes to PsAMADH2 and for enzyme activity, whereas N162, near catalytic C294, contributes mainly to the enzyme’s catalytic rate. Inside the substrate cavity, W170 and Y163, and, to a certain extent, L166 and M167 probably preserve the optimal overall geometry of the substrate channel that allows for the appropriate orientation of the substrate. Unconserved W288 appears to affect the affinity of the enzyme for the substrate amino group through control of the substrate channel diameter without affecting the reaction rate. Therefore, W288 may be a key determinant of the differences in substrate specificity found among plant AMADH isoforms when they interact with naturally occurring substrates such as 3-aminopropionaldehyde and 4-aminobutyraldehyde.
-
structural and functional characterization of plant Aminoaldehyde dehydrogenase from pisum sativum with a broad specificity for natural and synthetic Aminoaldehydes
Journal of Molecular Biology, 2010Co-Authors: Martina Tylichová, David Kopecny, Solange Moréra, Pierre Briozzo, Rene Lenobel, Jacques Snegaroff, Marek ŠebelaAbstract:Abstract Aminoaldehyde dehydrogenases (AMADHs, EC 1.2.1.19) belong to the large aldehyde dehydrogenase (ALDH) superfamily, namely, the ALDH9 family. They oxidize polyamine-derived ω-Aminoaldehydes to the corresponding ω-amino acids. Here, we report the first X-ray structures of plant AMADHs: two isoenzymes, PsAMADH1 and PsAMADH2, from Pisum sativum in complex with β-nicotinamide adenine dinucleotide (NAD+) at 2.4 and 2.15 A resolution, respectively. Both recombinant proteins are dimeric and, similarly to other ALDHs, each monomer is composed of an oligomerization domain, a coenzyme binding domain and a catalytic domain. Each subunit binds NAD+ as a coenzyme, contains a solvent-accessible C-terminal peroxisomal targeting signal (type 1) and a cation bound in the cavity close to the NAD+ binding site. While the NAD+ binding mode is classical for PsAMADH2, that for PsAMADH1 is unusual among ALDHs. A glycerol molecule occupies the substrate binding site and mimics a bound substrate. Structural analysis and substrate specificity study of both isoenzymes in combination with data published previously on other ALDH9 family members show that the established categorization of such enzymes into distinct groups based on substrate specificity is no more appropriate, because many of them seem capable of oxidizing a large spectrum of Aminoaldehyde substrates. PsAMADH1 and PsAMADH2 can oxidize N,N,N-trimethyl-4-aminobutyraldehyde into γ-butyrobetaine, which is the carnitine precursor in animal cells. This activity highly suggests that in addition to their contribution to the formation of compatible osmolytes such as glycine betaine, β-alanine betaine and γ-aminobutyric acid, AMADHs might participate in carnitine biosynthesis in plants.
-
light microscopic localisation of Aminoaldehyde dehydrogenase activity in plant tissues using nitroblue tetrazolium based staining method
Plant Physiology and Biochemistry, 2001Co-Authors: Marek Šebela, Lenka Luhova, Frantisek Aune, Pe Galuszka, Anna Radova, Pavel PecAbstract:We have recently isolated pea Aminoaldehyde dehydrogenase (AMADH, EC 1.2.1.-) and showed that it oxidises various ω-Aminoaldehydes, but not elementary aldehydes and betaine aldehyde. Now a simple method for specific staining in polyacrylamide gels was optimised enabling us to localise AMADH activity in plant tissues. When phenazine methosulphate (PMS) was used as a mediator, AMADH in native PAGE gels readily reduced thiazolyl blue (MTT) or nitroblue tetrazolium (NBT) producing the corresponding coloured formazans. Using NBT-based staining solution, AMADH activity was localised in cross sections of the root, hypocotyl, epicotyl and shoot apex of 7-d-old etiolated pea seedlings. We followed a histochemical approach for the enzyme localisation in order to visualise tissues where the Aminoaldehydes formed by the amine oxidase reaction are probably metabolised. In the root and hypocotyl, activity staining was most intense in cells belonging to the pericycle and endodermis. Weaker staining (namely in the root) was observed in the vascular cambium. In both epicotyl and shoot apex, the major part of AMADH activity appeared in vascular cambium cells. The violet formazan production was also observed in the pericycle and endodermis, but the staining intensity was lower. Pea amine oxidase, which produces naturally occurring Aminoaldehydes as potential AMADH substrates, is known as an apoplastic enzyme associated with tissues undergoing lignification (xylem, sclerenchyma) and wall stiffening (epidermis). Biological implications resulting from the localisation of both probably co-operating enzymes are discussed.
-
a study on the reactions of plant copper amine oxidase with c3 and c4 aliphatic diamines
Archives of Biochemistry and Biophysics, 2000Co-Authors: Marek Šebela, Ivo Frebort, Karel Lemr, Frantisek Brauner, Pavel PecAbstract:Abstract The paper reports a study on the reactions of grass pea (Lathyrus sativus) amine oxidase (GPAO) with several aliphatic diamines. The influence of the chain length and of unsaturations in the molecules was examined. Kinetic measurements confirmed that trans-, i.e., (E)-2-butene-1,4-diamine (TDABE) and cis-, i.e., (Z)-2-butene-1,4-diamine (CDABE) could be classified as good substrates. Propane-1,3-diamine (DAP) and propene-1,3-diamine (DAPE) were only weakly oxidized, whereas 1,3-diamino-2-propanol (DAPL) was not utilized as a substrate. Contrary to the inactivator 2-butyne-1,4-diamine (DABI), DAPE was shown to be only a competitive inhibitor. DAP itself did not inhibit the catalytic activity. Irreversible inhibition of the activity occurred only after the incubation of GPAO with DABI; other diamines were without this effect. Differential pulse polarography and chromatofocusing confirmed that the Aminoaldehyde product of DABI oxidation binds to the enzyme. Activity assay of pea Aminoaldehyde dehydrogenase enabled us to detect the products of the oxidation of TDABE, CDABE, and DAP by GPAO. As the product of DAP oxidation, 3-aminopropanal (APAL) was detected by mass spectrometry and confirmed to be a potent noncompetitive inhibitor of GPAO. The absorption changes that occurred in the course of the reaction of GPAO with the diamines were investigated using rapid-scanning spectrophotometry. DABI, TDABE, CDABE, DAP, and DAPE reacted with GPAO providing characteristic maxima of the Cu(I)-semiquinolamine species that is formed in the catalytic cycle. The results presented here confirm that with the exception of DAPL, all the studied diamines could be classified as GPAO substrates, but only DABI can be considered as a mechanism-based inhibitor.