Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology
Characterisation of a homogeneous plant aminoaldehyde dehydrogenase
Introduction
Polyamines have been proposed as regulatory substances involved in growth, cell division and differentiation [1], [2]. Catabolic regulation of the polyamine level in tissues is mediated by quinoprotein Cu-amine oxidases (Cu-AOs; EC 1.4.3.6) and flavoprotein FAD-polyamine oxidases (PAOs; EC 1.5.3.-) [3]. Plant Cu-AOs catalyse the oxidative deamination of di- and polyamine substrates, such as putrescine and spermidine, resulting in the formation of 4-aminobutyraldehyde (ABAL) and 4-(3-aminopropyl)aminobutyraldehyde (APBAL), respectively, along with the release of ammonia and H2O2 [4]. Several plant Cu-AOs have been shown to convert propane-1,3-diamine into 3-aminopropionaldehyde (APAL) [5], [6]. On the contrary, plant PAOs catalyse the oxidation of spermidine and spermine. The oxidative cleavage of the substrates bring about formation of ABAL and APBAL, respectively, propane-1,3-diamine and H2O2 [1].
In plants, the aminoaldehydes are further metabolised by the activity of NAD-dependent aminoaldehyde dehydrogenases (AMADHs, EC 1.2.1.19 or 1.2.1.54). The AMADH activities have been found in legumes and grasses [7], however, the enzymes have not yet been purified to homogeneity and extensively characterised [8], [9], [10], [11]. The best substrates of the enzymes, e.g. APAL, ABAL and 4-guanidinobutyraldehyde (GBAL), are oxidised to the respective ω-amino acids [8], [9], [10], [11].
Some higher plants accumulate betaine (glycine betaine) in response to salt stress or water deficit. Betaine is formed by two-step oxidation of choline via betaine aldehyde. The second step is catalysed by a chloroplastic NAD-dependent betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8) [12]. So far, there are reports on the isolation of plant BADH genes from spinach [13], sugar beet [14] and barley [15]. The BADH gene from sugar beet has been expressed in tobacco. The enzyme formed in the transformed leaves additionally oxidised APAL and ABAL [8]. Recently, the crystal structure of BADH from cod liver has been published [16].
Amino acids and biogenic amines can be utilised by bacterial cells as both a carbon and a nitrogen source [17]. In one of such metabolic pathways, putrescine is converted into succinate via 4-aminobutyric acid (GABA) [18]. NAD-dependent dehydrogenases involved in these reactions catalyse the oxidative conversion of ABAL to GABA or GBAL to 4-guanidinobutyrate [19]. The enzymes from Escherichia coli [20], Pseudomonas putida [19] and Hafnia alvei [21] were only partially purified and characterised, similarly as APAL dehydrogenase involved in the metabolism of norspermidine and propane-1,3-diamine in Arthrobacter TMP-1 [22].
Non-specific aldehyde dehydrogenases (ALDHs, EC 1.2.1.3 or EC 1.2.1.5) have been purified to homogeneity from several mammalian sources. The substrates of these enzymes range from elementary aldehydes to the aldehyde metabolites of naturally occurring compounds [23]. The cytoplasmic E3 isoenzyme of human liver ALDH efficiently catalyses the conversion of ABAL to GABA [24]. Other ALDHs metabolising ABAL were recently purified from human [25], rat [26] and bovine brain [27]. The mentioned E3 isoenzyme of human liver ALDH has been shown to have even a BADH activity [28]. Primary structures of non-specific mammalian ALDHs show large similarities to those of plant and mammalian BADHs, including absolute conservation of the active site residues [13].
First, the purification procedure, which allows to achieve a homogeneous preparation of a plant AMADH, is described in the presented paper. The enzyme was isolated from etiolated pea seedlings and it was found to be a tetramer in the native state. Molecular and kinetic properties of pea AMADH were extensively characterised. The N-terminal amino acid sequence of the enzyme shows a high degree of homology when compared with sequences of plant BADHs. Physiological implications of the obtained results are discussed.
Section snippets
Chemicals
APAL diethylacetal was obtained from Acros (Geel, Belgium), ABAL and aminoacetaldehyde diethylacetals were from Aldrich (Milwaukee, WI, USA). Betaine aldehyde hydrochloride was from Sigma (St. Louis, MA, USA). GBAL diethylacetal was synthesised from ABAL diethylacetal [29]. By the application of the same procedure, 3-guanidinopropionaldehyde diethylacetal and 4-guanidino-2-hydroxybutyraldehyde diethylacetal were prepared from the respective ω-aminoaldehydes. 4-Amino-2-hydroxybutyraldehyde
Purification of pea seedling AMADH
Prior to isolation, AMADH activity was examined in extracts from pea seedlings germinated in the dark for 1–10 days. In the whole seedlings after removal of roots, the enzyme was barely detectable on day 3 and the activity continually increased until day 6. Then it decreased. For the dehydrogenase purification, 6-day-old fresh seedlings were found to be optimal concerning the activity content as well as quality of the plant material. Frozen seedlings were not used since the total amount of
Discussion
Etiolated seedlings of legumes are known as good sources of highly active Cu-AOs localised in the cell wall [48]. The best substrates of the enzyme from pea seedlings, naturally occurring polyamines putrescine, cadaverine, agmatine and spermidine, are oxidatively deaminated to ABAL, AVAL, GBAL and APBAL, respectively [48]. These aminoaldehydes are further converted into the respective ω-amino acids [49]. The occurrence of a NAD-dependent ABAL (pyrroline) dehydrogenase in pea and oat seedlings
Acknowledgements
The authors thank Prof. Macholán from the Department of Biochemistry, Faculty of Science, Masaryk University, Brno, for kindly supplying chemicals, helpful discussions and sincere encouragement throughout this study. The work was supported by the Grants 203/99/D048 of the Grant Agency of the Czech Republic and ME 153/1998 from the Ministry of Education, Czech Republic.
References (59)
- et al.
Plant Sci.
(1999) - et al.
J. Ferment. Bioeng.
(1995) - et al.
Phytochemistry
(1995) Phytochemistry
(1996)- et al.
Phytochemistry
(1997) - et al.
Phytochemistry
(1995) - et al.
Arch. Biochem. Biophys.
(1989) FEMS Microbiol. Rev.
(1992)- et al.
Biochim. Biophys. Acta
(1986) - et al.
Biochimie
(1987)
J. Biol. Chem.
J. Biol. Chem.
Biochem. Biophys. Res. Commun.
Biochim. Biophys. Acta
Biochim. Biophys. Acta
Phytochemistry
J. Plant Physiol.
Phytochemistry
Anal. Biochem.
Biochim. Biophys. Acta
Phytochemistry
Gene
Biosens. Bioelectron.
Arch. Biochem. Biophys.
Phytochemistry
Phytochemistry
Physiol. Plant
Plant Growth Regul.
Cited by (58)
Silicon augments salt tolerance through modulation of polyamine and GABA metabolism in two indica rice (Oryza sativa L.) cultivars
2021, Plant Physiology and BiochemistryCitation Excerpt :Enzyme activity was expressed in pmol D-pyrroline min−1 mg−1 protein using an extinction coefficient of 1.863103 mol−1 cm−1. AMADH activity was estimated spectrophotometrically by monitoring the production of NADH (Šebela et al., 2000a). Enzyme activity was expressed as nmol NADH min−1 mg−1 protein.
Exogenous calcium chloride (CaCl<inf>2</inf>) promotes γ-aminobutyric acid (GABA) accumulation in fresh-cut pears
2021, Postharvest Biology and TechnologyGenomic organisation, activity and distribution analysis of the microbial putrescine oxidase degradation pathway
2013, Systematic and Applied MicrobiologyCitation Excerpt :Support for this pathway can draw on analysis of plant response to a range of stressors where oxidases were induced with the accumulation of protective 4-aminobutyrate [72]. While aminoaldehyde dehydrogenases are widely accepted as the enzymes chiefly responsible for the conversion of aminobutyraldehyde to aminobutyrate, few have been explicitly identified and characterised [35,58]. Multiple gene homologues, differing subcellular locations and broad substrate specificity have hindered an explicit identification of dehydrogenase catalytic activity in the overall catabolic process.
Genome-wide identification and analysis of the aldehyde dehydrogenase (ALDH) gene superfamily in apple (Malus×domestica Borkh.)
2013, Plant Physiology and BiochemistryPlant ALDH10 family identifying critical residues for substrate specificity and trapping a thiohemiacetal intermediate
2013, Journal of Biological ChemistryStructural determinants of substrate specificity in aldehyde dehydrogenases
2013, Chemico-Biological InteractionsCitation Excerpt :The residue at position 465 appears to be important for binding of BAL, as discussed below. Every biochemically characterized AMADH is able to efficiently oxidize APAL, ABAL, and TMABAL, but it has been found that not all of the ALDH10 enzymes can oxidize BAL [21,23,58,59]. Moreover, even those AMADH enzymes that can oxidize BAL exhibit a lower affinity for this substrate than for the other aminoaldehydes, as assessed by Km values [21,45,46,60].