Betaine Aldehyde Dehydrogenase Research Articles

Photosynthetic Adaptation to Salt Stress in Three-Color Leaves of a C4 Plant Amaranthus tricolor

We examined the photosynthetic adaptation mechanisms for salt stress in Amaranthus tricolor, which has leaves with green, yellow and red regions, in relation to the accumulation of glycinebetain e as osmoprotectants. The content of Chi, especially of Chi b in the red and yellow regions was 3~4% of that in the green region. The levels of Chi proteins such as LHCII, PSI and PSII were significantly lower than those in the green region. However, the contents of other photosynthetic proteins in these regions seem to be relatively high. We observed the net photosynthetic CO2 fixation activity in the red and yellow regions which was about 40% of that in the green region. Upon salt stress (0.3 M NaCl) for 5 d the levels of Chi, PSI, PSII, ribulose 1,5-bis phosphate carboxygenase and oxygenase, and the CO2 fixation rate in the green region decreased by about 20~35% whereas those in the non-green regions remained almost at the same levels. A. tricolor was found to accumulates glycinebetaine, betainealdehyde dehydrogenase and choline monooxygenase at similar levels in all three color regions and their contents increased upon salt stress. These results suggest that the low capacity of light harvesting in non-green regions would be favor of salt stress since the photosynthetic components in these regions were retained at relatively high levels under high salinity.

Characterization of an aldehyde dehydrogenase gene fragment from mung bean (Vigna radiata) using the polymerase chain reaction.

NAD(P)+-dependent aldehyde dehydrogenases of higher plants have not been studied well enough at the moment as compared to those of mammals. The betaine aldehyde dehydrogenase gene was obtained from rice (Nacamyra et al., 1997). There is some information on the structure of spinach, sugar beet, sorghum, burley cDNAs (Rhodes and Hanson, 1993; Wood et al., 1996), non-phOsphorylating glyceraldehyde 3-phosphate dehydrogenase cDNAs from pea and maize (Habenicht et al., 1994), maize RF2 protein cDNA (about 60% identity to mammalian mitochondrial aldehyde dehydrogenases) (Cui et al., 1996), tobacco mitochondrial aldehyde dehydrogenase cDNA (op den Camp and Kuhlemeier, 1997).KeywordsAldehyde DehydrogenaseBetaine Aldehyde DehydrogenaseAldehyde Dehydrogenase GeneMitochondrial Aldehyde DehydrogenaseBetaine Aldehyde Dehydrogenase GeneThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Structure and function of betaine aldehyde dehydrogenase. An enzyme within the multienzyme aldehyde dehydrogenase system.

Aldehyde and alcohol dehydrogenases (ALDHs and ADHs) are both highly multiple enzymes with many different forms in metabolically linked functional pathways. However, they are derived from separate protein families with distinct properties. Those of the vertebrate ADHs have been well characterized since long, and their different classes belong to the MDR protein family of known structures from several sources. However, the properties and multiplicity of the ALDHs have only recently been partly characterized. Presently, just three of the at least twelve different ALDH classes (Yoshida et al., 1998) are kn in three-dimensional structure (Liu et al., 1997; Steinmetz et al., 1997; Johansson et al.998), several have not been purified and characterized enzymatically, and little is known about their species or class variabilities.KeywordsAldehyde DehydrogenaseBetaine AldehydeSuccinic SemialdehydeBetaine Aldehyde DehydrogenaseCoenzyme BindingThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Accumulation of glycinebetaine and its synthesis from radioactive precursors under salt-stress in the cyanobacterium Aphanothece halophytica

Growth in salt-stressed (2.0 M NaCl) Aphanothece halophytica was initially delayed during the first two days of cultivation and eventually attained the same growth rate as the control (0.5 M NaCl) cells. Glycinebetaine accumulation increased slightly in control cells but a dramatic increase of glycinebetaine occurred in salt-stressed cells during a growth period of six days. There was no apparent increase in the synthesis of [14C] glycinebetaine in the control cells, in contrast to the marked increase in its synthesis in the salt-stressed cells. Increasing NaCl concentration in the growth medium induced both the accumulation and the synthesis of glycinebetaine. Time course experiments provided evidence that [14C] choline was first oxidized to [14C] betaine aldehyde which was further oxidized to [14C] glycinebetaine in A. halophytica. The supporting data for such a pathway were obtained from the presence of choline and betaine aldehyde dehydrogenase activities found in the membrane and cytoplasmic fractions, respectively. The activities of these two enzymes were also enhanced upon increasing NaCl concentration in the growth medium from 0.5 M to 2.0 M. Under this condition an increaseof approximately 1.5-fold was observed for choline dehydrogenase activity as compared to 2.5-fold for betaine aldehyde dehydrogenase activity, suggesting a preferable induction of the latter enzyme by salt stress. A. halophytica was able to utilize [14C] ethanolamine and [14C] glycine for the synthesis of [14C] glycinebetaine.

Evidence for mitochondrial localization of betaine aldehyde dehydrogenase in rat liver: purification, characterization, and comparison with human cytoplasmic E3 isozyme

Evidence for mitochondrial localization of betaine aldehyde dehydrogenase in rat liver: purification, characterization, and comparison with human cytoplasmic E3 isozyme

Isolation and molecular characterization of theSinorhizobium meliloti bet locus encoding glycine betaine biosynthesis

To cope with osmotic stress,Sinorhizobium meliloti accumulates organic compatible solutes such as glutamate, trehalose, N-acetylglutaminylglutamine amide, and the most potent osmoprotectant glycine betaine. In order to study the regulation of the glycine betaine biosynthetic pathway, a genetic and molecular analysis was performed. We have selected a Tn5 mutant ofS. meliloti which was deficient in choline dehydrogenase activity. The mutation was complemented using a genomic bank ofS. meliloti. Subcloning and DNA sequencing of a 8-6 kb region from the complemented plasmid showed four open reading frames with an original structural organization of thebet locus compared to that described inE. coli. (i) ThebetB and thebetA genes which encode a glycine betaine aldehyde dehydrogenase, and a choline dehydrogenase, respectively, are separated from thebetI gene (regulatory protein) by an additional gene namedbetC. The BetC protein shares about 30% identity with various sulphatases and is involved in the conversion of choline-O-sulphate into choline. Choline-O-sulphate is used as an osmoprotectant, or as a carbon or sulphur source and this utilization is dependent on a functionalbet locus. (ii) No sequence homologous tobetT (encoding a high-affinity choline transport system inE. coli) was found in the vicinity of thebet locus. (iii) ThebetB and thebetA genes, as well as thebetI and thebetC genes are, respectively, separated by 211 and 167 bp sequences containing inverted repeats. Southern blot analysis indicated that thebet locus is located on the chromosome, and not on the megaplasmids.

Structure of betaine aldehyde dehydrogenase at 2.1 A resolution.

The three-dimensional structure of betaine aldehyde dehydrogenase, the most abundant aldehyde dehydrogenase (ALDH) of cod liver, has been determined at 2.1 A resolution by the X-ray crystallographic method of molecular replacement. This enzyme represents a novel structure of the highly multiple ALDH, with at least 12 distinct classes in humans. This betaine ALDH of class 9 is different from the two recently determined ALDH structures (classes 2 and 3). Like these, the betaine ALDH structure has three domains, one coenzyme binding domain, one catalytic domain, and one oligomerization domain. Crystals grown in the presence or absence of NAD+ have very similar structures and no significant conformational change occurs upon coenzyme binding. This is probably due to the tight interactions between domains within the subunit and between subunits in the tetramer. The oligomerization domains link the catalytic domains together into two 20-stranded pleated sheet structures. The overall structure is similar to that of the tetrameric bovine class 2 and dimeric rat class 3 ALDH, but the coenzyme binding with the nicotinamide in anti conformation, resembles that of class 2 rather than of class 3.

Presence of a gene encoding choline sulfatase in Sinorhizobium meliloti bet operon: Choline- O -sulfate is metabolized into glycine betaine

Glycine betaine is a potent osmoprotectant accumulated by Sinorhizobium meliloti to cope with osmotic stress. The biosynthesis of glycine betaine from choline is encoded by an operon of four genes, betICBA, as determined by sequence and mutant analysis. The betI and betC genes are separated by an intergenic region containing a 130-bp mosaic element that also is present between the betB and betA genes. In addition to the genes encoding a presumed regulatory protein (betI), the betaine aldehyde dehydrogenase (betB), and the choline dehydrogenase (betA) enzymes also found in Escherichia coli, a new gene (betC) was identified as encoding a choline sulfatase catalyzing the conversion of choline-O-sulfate and, at a lower rate, phosphorylcholine, into choline. Choline sulfatase activity was absent from betC but not from betB mutants and was shown to be induced indifferently by choline or choline-O-sulfate as were the other enzymes of the pathway. Unlike what has been shown in other bacteria and plants, choline-O-sulfate is not used as an osmoprotectant per se in S. meliloti, but is metabolized into glycine betaine. S. meliloti also can use this compound as the sole carbon, nitrogen, and sulfur source for growth and that depends on a functional bet locus. In conclusion, choline-O-sulfate and phosphorylcholine, which are found in higher plants and fungi, appear to be substrates for glycine betaine biosynthesis in S. meliloti.

Molecular characterization of two genes encoding betaine aldehyde dehydrogenase from amaranth. Expression in leaves under short-term exposure to osmotic stress or abscisic acid.

A genomic clone (ahybadh4) and a cDNA (ahybadh17) both encoding betaine aldehyde dehydrogenase (BADH; EC 1.2.1.8) were isolated from the plant Amaranthus hypochondriacus L. The ahybadh4 gene extends 9 kilobases (kb) containing 15 exons with an open reading frame (ORF) of 501 amino acids (aa), a 1.3kb 5' untranslated region (UTR) and a 3' UTR of 0.3kb. The ahybadh17 cDNA encodes a BADH isoform of 500aa which contains 10aa substitutions with respect to AHYBADH4. Both encoded proteins share 98% identity at the amino acid level. Comparison of amaranth BADHs with other reported sequences showed high similarity. Analysis of ahybadh17 expression in amaranth leaves showed that mRNA and BADH protein are present in non-treated amaranth leaves and both transiently increased under short-term exposure to abscisic acid (ABA) and osmotic stress treatments.

Genetic Structure of thebphGGene Encoding 2-Hydroxymuconic Semialdehyde Dehydrogenase ofAchromobacter xylosoxidansKF701

2-Hydroxymuconic semialdehyde dehydrogenase catalyzes the conversion of 2-hydroxymuconic semialdehyde (HMS) to an enol form of 4-oxalocrotonate which is a step in the catecholmeta-cleavage pathway. AbphGgene encoding HMS dehydrogenase ofA. xylosoxidansKF701, a soil bacterium degrading biphenyl, was identified at between catechol 2,3-dioxygenase gene and HMS hydrolase gene, and its sequence was analyzed. An open reading frame (ORF) corresponding tobphGgene was consisted of 1461 nucleotides with ATG initiation codon and TGA termination codon. The ORF exhibited 66% of G + C content, and a putative ribosome-binding sequence, AGAGA, was identified at about 10 nucleotides upstream initiation codon of thebphGgene. ThebphGgene can encode a polypeptide of molecular weight 52 kDa containing 486 amino acid residues. A deduced amino acid sequence of HMS dehydrogenase encoded inbphGgene fromA. xylosoxidansKF701 exhibited the highest 94% homology with that of corresponding enzyme encoded inxylGfromP. putidamt-2, 63% to 90% homology with those of other reported HMS dehydrogenases, and 29% to 42% homology with those of betaine aldehyde dehydrogenase, 5-carboxy-HMS dehydrogenase, aldehyde dehydrogenase, indole-3-acetaldehyde dehydrogenase, succinic semialdehyde dehydrogenase, methylmalonate semialdehyde dehydrogenase, and succinylglutamate 5-semialdehyde dehydrogenase. From an alignment of amino acid sequence of HMS dehydrogenase fromA. xylosoxidansKF701 with other reported dehydrogenases, putative cofactor NAD+-binding regions and catalytic residues were identified.

Direct fermentation of 2-keto-l-gulonic acid in recombinantGluconobacter oxydans

We isolated Gluconobacter oxydans T-100 that had an activity to produce 2-KLGA from D-sorbitol; however, the yield of 2-KLGA was quite insufficient. Therefore, enzymes involved in the biosynthesis of L-sorbosone and 2-KLGA, L-sorbose dehydrogenase (SDH) and L-sorbosone dehydrogenase (SNDH), respectively, were purified from G. oxydans T-100. A genomic library of G. oxydans T-100 was screened to clone both genes for SDH and SNDH based on their amino acid sequences. SNDH and SDH were encoded in sequential open reading frames with 1497 and 1596 nucleotides, respectively, which were verified by the expression in Escherichia coli. The amino acid sequence of SDH and SNDH showed close similarity with E. coli choline dehydrogenase (CDH) and betaine-aldehyde dehydrogenase (BADH), respectively, which cooperatively play a key role for conferring osmotic tolerance. Because the yield of 2-KLGA by G. oxydans introduced with the genes for SDH and SNDH were insufficient, replacement of the promoter with that of Escherichia coli tufB1 in combination with chemical mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine resulted in improvement of the production level.

Porcine kidney betaine aldehyde dehydrogenase: purification and properties

Significant betaine aldehyde dehydrogenase activity was found in porcine kidney. The enzyme was purified 320-fold with an overall recovery of 11%. It had a specific activity of 115.8 nkats/mg protein and proved to be homogeneous by SDS-PAGE with a subunit molecular mass of 52 kDa. IEF studies showed three bands with pI values of 5.74, 5.68 and 5.58, respectively. The enzyme was stable in a pH range between 5.0 and 10.0 and the optimum pH was 9.5. The reaction is highly specific for NAD + and betaine aldehyde, although acetaldehyde, butyraldehyde and glyceraldehyde can be used. Estimated values of K m at pH 8.0 and 25°C were 127 μM for betaine aldehyde and 40 μM for NAD +. The reaction could not be reversed even at high glycine betaine concentrations. The enzyme was not activated by salts at high concentrations but it was salt tolerant—retaining 50% of maximal activity at 1.0 M K + and Na +. It is inferred that salt tolerance is an essential property for an enzyme participating in the cellular synthesis of an osmoprotectant. Proline, glycerol, sucrose and mannitol had a little effect on the enzyme activity while glycine betaine had an inhibitory effect.

Genomic cloning and sequencing of betaine aldehyde dehydrogenase gene in spinach

A genornic library derived from leaves of spinach was constructed with the λGeml1-BamHI Arms as the vector. The library was screened using thc BADH cDNA of mountiain spinach as a probe and six positive clones were obtained through three rounds of screening. One of the positive clones named D, which was hybridized with the 5’600 bp fragment of mountain spinach BADH cDNA, was sclcctrd and further analyzed. The size of the insert in clone D was about 12 kb. 8 856 nucleotides of the insert were sequenced which contained 2 459 nucleotides of 5′ noncoding region, 6 111 nucleotides of the complete sequence of the BADH gene, and 286 nuclrotides of a 3′ noncoding rcgion. The result of sequence analysis indicated that the BADH gene contained 14 introns and the junction scquenccs at splicing sites followed the GT-AG rule basically.

Expression of betaine aldehyde dehydrogenase gene and salinity tolerance in rice transgenic plants

Betaine as one of osmolytes plays an important role in osmoregulation of most high plants. Betaine aldehyde dehydrogenase (BADH) is the second enzyme involved in betaine biosynthesis. The BADH gene from a halophite,Atriplex hortensis, was transformed into rice cultivars by bombarment method. Totally 192 transgenic rice plants were obtained and most of them had higher salt tolerance than controls. Among transgenic plants transplanted in the saline pool containing 0.5% NaCl in a greenhouse, 22 survived. 13 of which set seeds, and the frequency of seed setting was very low, only 10%. But the controls could not grow under the same condition. The results of BADH activity assay and Northern blot showed that the BADH gene was integrated into chromosomes of transgenic plants and expressed.

Glycine betaine aldehyde dehydrogenase from Bacillus subtilis: characterization of an enzyme required for the synthesis of the osmoprotectant glycine betaine.

Production of the compatible solute glycine betaine from its precursors choline or glycine betaine aldehyde confers a considerable level of tolerance against high osmolarity stress to the soil bacterium Bacillus subtilis. The glycine betaine aldehyde dehydrogenase GbsA is an integral part of the osmoregulatory glycine betaine synthesis pathway. We strongly overproduced this enzyme in an Escherichia coli strain that expressed a plasmid-encoded gbsA gene under T7φ10 control. The recombinant GbsA protein was purified 23-fold to apparent homogeneity by fractionated ammonium sulfate precipitation, ion-exchange chromatography on Q-Sepharose, and subsequent hydrophobic interaction chromatography on phenyl-Sepharose. Molecular sieving through Superose 12 and sedimentation centrifugation through a glycerol gradient suggested that the native enzyme is a homodimer with 53.7-kDa subunits. The enzyme was specific for glycine betaine aldehyde and could use both NAD+ and NADP+ as cofactors, but NAD+ was strongly preferred. A kinetic analysis of the GbsA-mediated oxidation of glycine betaine aldehyde to glycine betaine revealed Km values of 125 microM and 143 microM for its substrates glycine betaine aldehyde and NAD+, respectively. Low concentrations of salts stimulated the GbsA activity, and the enzyme was highly tolerant of high ionic conditions. Even in the presence of 2.4 M KCl, 88% of the initial enzymatic activity was maintained. B. subtilis synthesizes high levels of proline when grown at high osmolarity, and the presence of this amino acid strongly stimulated the GbsA activity in vitro. The enzyme was stimulated by moderate concentrations of glycine betaine, and its activity was highly tolerant against molar concentrations of this osmolyte. The high salt tolerance and its resistance to its own reaction product are essential features of the GbsA enzyme and ensure that B. subtilis can produce high levels of the compatible solute glycine betaine under conditions of high osmolarity stress.

Substrate inhibition by betaine aldehyde of betaine aldehyde dehydrogenase from leaves of Amaranthus hypochondriacus L.

We have previously proposed that at low substrate concentrations betaine aldehyde dehydrogenase follows an irreversible Iso Ordered Bi Bi Steady State kinetic mechanism with NAD + as the leading substrate [E.M. Valenzuela-Soto and R.A. Muñoz-Clares, J. Biol. Chem. 268 (1993) 23818–23823]. To further the understanding of this enzyme, we have studied the kinetics at high substrate concentrations. Betaine aldehyde at concentrations above 500 μM behaves as a non-competitive inhibitor against NAD +, with downward-curved slope and intercept replots. Double-inhibition studies, using NADH as the second inhibitor, show the formation of the abortive ternary complex enzyme·NADH·betaine aldehyde, from which NADH may escape at a finite rate, accounting for the nonlinear Dixon plots obtained for both inhibitors. In addition, the binary complex enzyme·betaine aldehyde may give rise to a slower alternative route of reaction, which, under our experimental conditions, was observed at NAD + concentrations above 1 mM, where double-reciprocal plots of initial velocity against [NAD +] and Dixon plots of 1/ v against [NADH] were concave downward. In contrast with other aldehyde dehydrogenases, no `substrate activation' by the aldehyde was observed under several conditions, which is consistent with the alternative route of reaction being slower than the route which operates at low substrate concentrations. Taken together, our results are consistent with the partial inhibition by high betaine aldehyde concentrations resulting from an irreversible Iso Random Steady State mechanism with a preferential route of reaction. Eventually, at very high betaine aldehyde concentrations, the kinetic mechanism may change to an apparent Ping Pong.

Transgenically Expressed Betaine Aldehyde Dehydrogenase Efficiently Catalyzes Oxidation of Dimethylsulfoniopropionaldehyde and [omega]-Aminoaldehydes.

Tobacco (Nicotianum tabacum L.) plants engineered to express a sugar beet (Beta vulgaris L.) betaine aldehyde dehydrogenase (BADH) cDNA acquired not only BADH activity, but also three other aldehyde dehydrogenase activities (those measured with 3-dimethylsulfoniopropionaldehyde, 3-aminopropionaldehyde, and 4-aminobutyraldehyde, all of which are natural products). This shows that BADH is not, as believed up to now, a substrate-specific enzyme and that its role may not be limited to glycine betaine synthesis.

Salt tolerance in two gramineae sp. Panicum maximum and Eleusine coracana: Glycinebetaine accumulation and expression of betaine aldehyde dehydrogenase mRNA

Two gramineae species, Panicum maximum and Eleusine coracana, were exposed to salinity stress and leaf water status, solute concentrations in cell sap, levels of betaine aldehyde dehydrogenase (BADH) and BADH mRNA were measured. Panicum maximum was able to maintain a high turgor and high relative water content at low leaf water potentials, and this was associated with the greater capacity for osmotic adjustment. Major osmotica in Panicum maximum leaves were Na+, Cl−, sugar and glycinebetaine, and these solutes were increased by salinity. Na+ and glycinebetaine concentrations in Eleusine coracana leaves also were increased by salt stress, but these solutes were in significantly lower concentrations than those measured in Panicum maximum. BADH enzyme activity and BADH mRNA levels in Panicum maximum leaves were both increased by salt stress, and their expression coincided with the observed betaine accumulation. Although the addition of abscisic acid (ABA) to leaf disks of Panicum maximum plants also increased BADH mRNA levels, these were smaller than those observed in NaCl treated leaves.

Possible Involvement of Greening in Cell Growth of Suaeda japonica under Salt Stress

Suaeda japonica, a member of the family Chenopodiaceae, is a halophyte that grows on the shores of the Ariake Sea in Japan. Using yellowish and selected green callus tissues of S. japonica, we examined the correlation between cell growth and glycinebetaine content, as well as the activity of betaine aldehyde dehydrogenase (BADH), under salt stress. Although the growth of yellowish callus tissues was markedly inhibited by 0.1 M NaCl, that of green callus tissues was not. However, in 0.3 M NaCl, the fresh weight of green callus tissues was decreased by 15%. Both the endogenous level of glycinebetaine and the activity of BADH in yellowish callus tissues were very low, but green callus tissues contained very high levels of glycinebetaine and high BADH activity. The activity in green callus increased significantly with increases in the concentration of NaCl. The increase in levels of Chl was also observed, but the increase occurred earlier than that in the level of glycinebetaine. Functional development of chloroplasts (an increase in the level of Chl) seems to be involved in the increase in the activity of BADH, and the resultant increase in levels of glycinebetaine seems to facilitate callus growth under salt stress.

Betaine-Aldehyde Dehydrogenase from Amaranth Leaves Efficiently Catalyzes the NAD-Dependent Oxidation of Dimethylsulfoniopropionaldehyde to Dimethylsulfoniopropionate

Many flowering plants accumulate the compatible osmolyte glycine betaine in response to osmotic stress, in certain cases together with its sulfonium analog 3-dimethylsulfoniopropionate (DMSP). Compared to glycine betaine, this DMSP accumulation appears to be an evolutionary novelty. The final step in the synthesis of glycine betaine and DMSP is oxidation of the corresponding aldehyde, betaine aldehyde or 3-dimethylsulfoniopropionaldehyde (DMSPald). Leaves of amaranth (Amaranthus hypochondriacusL.) accumulate glycine betaine but do not produce detectable amounts of DMSP. These leaves contain a betaine-aldehyde dehydrogenase (BADH) that catalyzes the final step in glycine betaine synthesis. Here we report that this enzyme efficiently catalyzes the oxidation of DMSPald. On the basis ofVmax/Kmvalues, DMSPald is a better substrate for amaranth BADH than betaine aldehyde itself. The kinetic mechanism followed by amaranth BADH at low concentrations of DMSPald is similar to that with betaine aldehyde; as determined from initial velocity, product, dead-end, and substrate inhibition studies, it is a steady-state bi bi with ordered addition of substrates and random release of products. Like betaine aldehyde, DMSPald is inhibitory at high concentrations, at which a slower alternate route of substrate addition takes place. Our results indicate that the evolution of DMSP biosynthesis in flowering plants could have been facile in that it required no new aldehyde dehydrogenase; BADH may simply have been recruited for a novel function.