Characterisation of a homogeneous plant aminoaldehyde dehydrogenase

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Abstract

According to our knowledge, this is the first purification method developed, enabling isolation of a homogeneous aminoaldehyde dehydrogenase (AMADH) from etiolated pea seedlings. The procedure involved initial purification with precipitants followed by three low pressure chromatographic steps. Partially purified enzyme was further subjected to fast protein liquid chromatography on a Mono Q column and to affinity–interaction chromatography on 5′-AMP Sepharose. Purity of the final enzyme preparation was checked by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and chromatofocusing. Pea AMADH exists as a tetramer of 230 kDa in the native state, a molecular mass of one subunit was determined as 57 kDa. The enzyme was found to be an acidic protein with pI 5.4. AMADH showed a broad substrate specificity utilising various aminoaldehydes (C3–C6) as substrates. The best substrate of pea AMADH was 3-aminopropionaldehyde, the enzyme also efficiently oxidised 4-aminobutyraldehyde and ω-guanidinoanalogues of the aminoaldehydes. Pea AMADH was inhibited by SH reagents, several elementary aldehydes and metal-binding agents. Although AMADH did not oxidise betaine aldehyde at all, the N-terminal amino acid sequence of the enzyme shows a high degree of homology with those of plant betaine aldehyde dehydrogenases (BADHs) of spinach, sugar beet and amaranth. Several conserved amino acids were found in comparison with BADH from cod liver of known crystal structure.

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.

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