Publications

• Higher activity of an aldehyde oxidase in the auxin-overproducing superroot1 mutant of Arabidopsis thaliana.

  Seo M., Akaba S., Oritani T., Delarue M., Bellini C., Caboche M., Koshiba T.

  Aldehyde oxidase (AO; EC 1.2.3.1) activity was measured in seedlings of wild type or an auxin-overproducing mutant, superroot1 (sur1), of Arabidopsis thaliana. Activity staining for AO after native polyacrylamide gel electrophoresis separation of seedling extracts revealed that there were three ma ... >> More

  Aldehyde oxidase (AO; EC 1.2.3.1) activity was measured in seedlings of wild type or an auxin-overproducing mutant, superroot1 (sur1), of Arabidopsis thaliana. Activity staining for AO after native polyacrylamide gel electrophoresis separation of seedling extracts revealed that there were three major bands with AO activity (AO1-3) in wild-type and mutant seedlings. One of them (AO1) had a higher substrate preference for indole-3-aldehyde. This AO activity was significantly higher in sur1 mutant seedlings than in the wild type. The difference in activity was most apparent 7 d after germination, the same time required for the appearance of the remarkable sur1 phenotype, which includes epinastic cotyledons, elongated hypocotyls, and enhanced root development. Higher activity was observed in the root and hypocotyl region of the mutant seedlings. We also assayed the indole-3-acetaldehyde oxidase activity in extracts by high-performance liquid chromatography detection of indole-3-acetic acid (IAA). The activity was about 5 times higher in the extract of the sur1 seedlings, indicating that AO1 also has a substrate preference for abscisic aldehyde. Treatment of the wild-type seedlings with picloram or IAA caused no significant increase in AO1 activity. This result suggested that the higher activity of AO1 in sur1 mutant seedlings was not induced by IAA accumulation and, thus, strongly supports the possible role of AO1 in IAA biosynthesis in Arabidopsis seedlings. << Less

  Plant Physiol. 116:687-693(1998) [PubMed] [EuropePMC]

• Functional expression of two Arabidopsis aldehyde oxidases in the yeast Pichia pastoris.

  Koiwai H., Akaba S., Seo M., Komano T., Koshiba T.

  To investigate the biochemical and enzymatic properties of two aldehyde oxidase (AO) isoforms of Arabidopsis thaliana, we expressed AAO1 and AAO2 cDNAs in a heterologous yeast (Pichia pastoris) system and successfully obtained the proteins in active forms. The expressed AAO1 and AAO2 proteins gave ... >> More

  To investigate the biochemical and enzymatic properties of two aldehyde oxidase (AO) isoforms of Arabidopsis thaliana, we expressed AAO1 and AAO2 cDNAs in a heterologous yeast (Pichia pastoris) system and successfully obtained the proteins in active forms. The expressed AAO1 and AAO2 proteins gave activity bands with the same mobilities on native gel electrophoresis and exhibited the same substrate preferences on zymograms with 8 aldehydes as those of AOalpha and AOgamma in Arabidopsis seedlings, respectively. Furthermore, anti-AAO1 and anti-AAO2 antibodies, which specifically recognize the seedling AOalpha and AOgamma, respectively, reacted with the AAO1 and AAO2 proteins produced in P. pastoris, respectively. These results indicate that these AO proteins are accurately produced in the yeast system, as in Arabidopsis seedlings. Using AO preparations from P. pastoris, the enzymatic properties of Arabidopsis AOalpha and AOgamma were investigated. AOalpha showed a relatively wide substrate specificity for 7 aldehydes tested, with high affinity to benzaldehyde and indole-3-aldehyde, while AOgamma could most efficiently oxidize naphthaldehyde. AOalpha was strongly inhibited by iodoacetate and KCN, while AOgamma was inhibited not only by iodoacetate and KCN but also by 2-mercaptethanol, dithiothreitol, menadion, and estradiol. AOalpha and AOgamma showed the highest activity at around 65 and 50 degrees C, respectively, and exhibited pH dependence around pH 8.0. These results indicate that the two AO isoforms in Arabidopsis seedlings have different enzymatic properties and may have different physiological roles in vivo. << Less

  J. Biochem. 127:659-664(2000) [PubMed] [EuropePMC]

• An aldehyde oxidase in developing seeds of Arabidopsis converts benzaldehyde to benzoic Acid.

  Ibdah M., Chen Y.-T., Wilkerson C.G., Pichersky E.

  Arabidopsis (Arabidopsis thaliana) siliques synthesize high levels of benzoic acid (BA), which is incorporated into several glucosinolate compounds. The origin of BA in the siliques has not yet been determined. Here, we show that siliques have higher levels of benzaldehyde (BD)-oxidizing activity ... >> More

  Arabidopsis (Arabidopsis thaliana) siliques synthesize high levels of benzoic acid (BA), which is incorporated into several glucosinolate compounds. The origin of BA in the siliques has not yet been determined. Here, we show that siliques have higher levels of benzaldehyde (BD)-oxidizing activity relative to leaves. The BD-oxidizing activity was purified from siliques in several chromatographic steps, and a 145-kD protein was identified as the enzyme most likely to possess this activity. The protein was trypsinized, and the sequence of the resulting peptides was determined by mass spectrometry, identifying it as the product of gene At1g04580, also designated as AAO4 (for ARABIDOPSIS ALDEHYDE OXIDASE4). AAO4 had previously been shown to be highly and specifically expressed in developing seeds, and its protein was shown to belong to a family of aldehyde oxidases. Here, we show that the AAO4 protein is an aldehyde oxidase that can use several substrates but that, among the substrates tested, has the lowest K(m) value (23 microm) with BD. AAO4 is able to oxidize BD without NAD(+), but its activity increases by 50% when this cofactor is added. The pH optimum of AAO4 is 7.0. Plants homozygous for a null allele in AAO4 showed a reduction of 30% to 45% in the total levels of BA in seeds as well as 7% to 9% and 32% to 38% decreases in the levels of 3-benzoyloxypropylglucosinolate and 4-benzoyloxybutylglucosinolate, respectively. Expressing AAO4 in Escherichia coli resulted in a 3-fold increase of BD-oxidizing activity in crude bacterial extracts over endogenous levels. These findings indicate that in Arabidopsis seeds, oxidation of BD contributes in part to the synthesis of BA. << Less

  Plant Physiol. 150:416-423(2009) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Molecular cloning and characterization of aldehyde oxidases in Arabidopsis thaliana.

  Sekimoto H., Seo M., Kawakami N., Komano T., Desloire S., Liotenberg S., Marion-Poll A., Caboche M., Kamiya Y., Koshiba T.

  Using degenerate primers designed by deduced amino acid sequences of known aldehyde oxidases (AO) from maize and bovine, two independent cDNA fragments were amplified by reverse transcription-polymerase chain reaction (PCR). The two corresponding full-length cDNAs (atAO-1 and atAO-2; 4,484 and 4,2 ... >> More

  Using degenerate primers designed by deduced amino acid sequences of known aldehyde oxidases (AO) from maize and bovine, two independent cDNA fragments were amplified by reverse transcription-polymerase chain reaction (PCR). The two corresponding full-length cDNAs (atAO-1 and atAO-2; 4,484 and 4,228 bp long, respectively) were cloned by screening the Arabidopsis cDNA library followed by rapid amplification of cDNA end-PCR. These cDNAs are highly homologous at both the nucleotide and amino acid sequence levels, and the deduced amino acid sequences showed high similarity with those of maize and tomato AOs. They contain consensus sequences for two iron-sulfur centers and a molybdenum cofactor (MoCo)-binding domain. In addition, another cDNA having a sequence similar to that of the cDNAs was screened (atAO-3; 3,049 bp), and a putative AO gene (AC002376) was reported on chromosome 1, which (atAO-4) was distinct from, but very similar to, the above three AOs. atAO-1, 2, 3, and 4 were physically mapped on chromosomes 5, 3, 2 and 1, respectively. These data indicate that there is an AO multigene family in Arabidopsis. atAO-1 protein was shown to be highly similar to one of the maize AOs in respect to a region thought to be involved in determination of substrate specificity, suggesting that they might encode a similar type of AO, which could efficiently oxidize indole-3-acetaldehyde to indole-3-acetic acid (IAA). atAO-1 and atAO-2 genes were expressed at higher levels in lower hypocotyls and roots of the wild-type seedlings, while atAO-3 was slightly higher in cotyledons and upper hypocotyls. The expression of atAO-1 was more abundant in the seedlings of an IAA overproducing mutant (superroot1; sur1) than in those of wild type. atAO-2 and atAO-3 transcripts were rather evenly distributed in these seedlings. A possible involvement of atAO genes in phytohormone biosynthesis in Arabidopsis is discussed. << Less

  Plant Cell Physiol. 39:433-442(1998) [PubMed] [EuropePMC]

• Production of homo- and hetero-dimeric isozymes from two aldehyde oxidase genes of Arabidopsis thaliana.

  Akaba S., Seo M., Dohmae N., Takio K., Sekimoto H., Kamiya Y., Furuya N., Komano T., Koshiba T.

  Polyclonal antibodies were raised against synthetic peptides or recombinant polypeptides encoded by Arabidopsis atAO-1 and atAO-2 cDNAs, which have sequences similar to maize and animal aldehyde oxidase (AO) cDNAs. Anti-atAO-1 antibodies recognized AOalpha and AObeta among the three isoforms, AOal ... >> More

  Polyclonal antibodies were raised against synthetic peptides or recombinant polypeptides encoded by Arabidopsis atAO-1 and atAO-2 cDNAs, which have sequences similar to maize and animal aldehyde oxidase (AO) cDNAs. Anti-atAO-1 antibodies recognized AOalpha and AObeta among the three isoforms, AOalpha, AObeta, and AOgamma, detected in Arabidopsis seedlings after native PAGE, while anti-atAO-2 antibodies reacted with AObeta and AOgamma. The polypeptide specifically recognized by each antibody was collected as the Protein-A/IgG/antigen complex. The 150- and 145-kDa polypeptides were purified by SDS-PAGE and digested with Achromobacter Protease I. From the amino acid sequences and molecular masses of the derivative peptides, it was revealed that the 150- and 145-kDa polypeptides were the products of atAO-1 and atAO-2, respectively. Molecular masses of the native forms of AOalpha, AObeta, and AOgamma were estimated as approximately 290-300 kDa. These results suggest that AOalpha and AOgamma are homodimers consisting of atAO-1 and atAO-2 products, respectively, and that AObeta is a heterodimer of the atAO-1 and atAO-2 products. << Less

  J. Biochem. 126:395-401(1999) [PubMed] [EuropePMC]

• Purification and properties of flavin- and molybdenum-containing aldehyde oxidase from coleoptiles of maize.

  Koshiba T., Saito E., Ono N., Yamamoto N., Sato M.

  Aldehyde oxidase (AO; EC 1.2.3.1) that could oxidize indole-3-acetaldehyde into indole-3-acetic acid was purified approximately 2000-fold from coleoptiles of 3-d-old maize (Zea mays L.) seedlings. The apparent molecular mass of the native enzyme was about 300 kD as estimated by gel-filtration colu ... >> More

  Aldehyde oxidase (AO; EC 1.2.3.1) that could oxidize indole-3-acetaldehyde into indole-3-acetic acid was purified approximately 2000-fold from coleoptiles of 3-d-old maize (Zea mays L.) seedlings. The apparent molecular mass of the native enzyme was about 300 kD as estimated by gel-filtration column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the enzyme was composed of 150-kD subunits. It contained flavin adenine dinucleotide, iron, and molybdenum as prosthetic groups and had absorption peaks in the visible region (300-600 nm). To our knowledge, this is the first demonstration of the presence of flavin adenine dinucleotide and metals in plant AO. Other aromatic aldehydes such as indole-3-aldehyde and benzaldehyde also served as good substrates, but N-methylnicotinamide, a good substrate for animal AO, was not oxidized. 2-Mercaptoethanol, p-chloromercu-ribenzoate, and iodoacetate partially inhibited the activity, but well-known inhibitors of animal AO, such as menadione and estradiol, caused no reduction in activity. These results indicate that, although maize AO is similar to animal enzymes in molecular mass and cofactor components, it differs in substrate specificity and susceptibility to inhibitors. Immunoblotting analysis with mouse polyclonal antibodies raised against the purified maize AO showed that the enzyme was relatively rich in the apical region of maize coleoptiles. The possible role of this enzyme is discussed in relation to phytohormone biosynthesis in plants. << Less

  Plant Physiol. 110:781-789(1996) [PubMed] [EuropePMC]

• An in Vitro System of Indole-3-Acetic Acid Formation from Tryptophan in Maize (Zea mays) Coleoptile Extracts.

  Koshiba T., Matsuyama H.

  The formation of a product from tryptophan that had the same retention time as that of authentic indole-3-acetic acid (IAA) on high performance liquid chromatography was detected in crude extracts of maize (Zea mays) coleoptiles. The product was identified as IAA by mass spectrometry. The IAA-form ... >> More

  The formation of a product from tryptophan that had the same retention time as that of authentic indole-3-acetic acid (IAA) on high performance liquid chromatography was detected in crude extracts of maize (Zea mays) coleoptiles. The product was identified as IAA by mass spectrometry. The IAA-forming activity was co-purified with an indole-3-acetaldehyde (IAAld) oxidase activity by chromatography on hydrophobic and gel filtration (GPC-100) columns. During purification, the IAA-forming activity, rather than that of IAAld oxidase, decreased; but when hemoprotein obtained from the same tissue was added, activity recovered to the same level as that of IAAld oxidase. The promotive activity of the hemoprotein was confirmed by the result that the activity coincided with amounts of the hemoprotein after GPC-100 column chromatography. The hemoprotein was characterized and identified as a cytosolic ascorbate peroxidase (T. Koshiba [1993] Plant Cell Physiol [in press]). The reaction of the IAA-forming activity was apparently one step from tryptophan. The activity was inhibited by 2-mercaptoethanol. The optimum temperature for the IAA-forming system as well as for the IAAld oxidase was 50 to 60[deg]C, and the acitivity at 30[deg]C was one-third to one-half of that at 60[deg]C. The system did not discriminate the L- and D-enantiomers of tryptophan. << Less

  Plant Physiol 102:1319-1324(1993) [PubMed] [EuropePMC]