Tryptophan Monooxygenase Research Articles

Characterization of maize polyamine oxidase

Some structural and biochemical characteristics of polyamine oxidase (PAO) purified from maize shoots have been examined. The enzyme has only alanine as N-terminal amino acid and its N-terminal sequence shows a significant degree of homology with tryptophan 2-monooxygenase from Pseudomonas syringae pv. savastanoi. The pH optimum for the stability of the native enzyme is 5, similar to that of the barley leaf enzyme. Calorimetric analysis shows a single two-state transition at pH 6 with T m 49.8°. At pH 5 the thermal stability is increased by more than 14°. Amine oxidation products, Δ 1-pyrroline and diazabicyclononane, are competitive inhibitors of PAO activity (apparent K i=400 and 100 μM respectively). Moreover these compounds improve the thermal stability of the enzyme. N 1-Acetylspermine, which is a good substrate for mammalian PAO, acts as a non-competitive inhibitor for the plant enzyme.

Cotranscription of genes encoding indoleacetic acid production in Pseudomonas syringae subsp. savastanoi.

Indoleacetic acid (IAA) production by the plant pathogen Pseudomonas syringae subsp. savastanoi is essential for tumor formation on olive and oleander. The bacterium produces IAA from tryptophan in reactions catalyzed by tryptophan monooxygenase and indoleacetamide hydrolase. The genetic determinants are, respectively, iaaM and iaaH. In oleander isolates, the genes encoding the IAA biosynthetic enzymes are located on a plasmid; in olive isolates, the genes occur on the chromosome. The IAA genes from the oleander isolate strain EW2009 are located within a 4-kilobase (kb) segment of the 52-kb plasmid pIAA1. Escherichia coli strains harboring a recombinant plasmid, pCJP3, which contains this 4-kb fragment, excreted IAA into culture media, and crude cell extracts had both tryptophan monooxygenase and indoleacetamide hydrolase activity. In vitro coupled transcription-translation of pCJP3 demonstrated that this fragment coded for proteins of 62 and 47 kilodaltons which correspond to tryptophan monooxygenase and indoleacetamide hydrolase, respectively. Expression of these genes was dependent upon a vector promoter in pCJP3. However, in the absence of a vector promoter, E. coli containing recombinant plasmids with additional pIAA1 DNA in front of iaaM had high levels of tryptophan monooxygenase. Northern (RNA) hybridization experiments verified that iaaM and iaaH are cotranscribed as a portion of a ca. 4- to 5-kb transcript in vivo. Southern hybridization experiments with IAA plasmids from different oleander strains of P. syringae subsp. savastanoi revealed that all IAA plasmids contained a region of at least 10 kb of homology, with the IAA genes at one end. Repetitive DNA and a copy of IS51 were found at the end of this region of homology.

Intracellular metabolism of biogenic amines in paraneurons.

Serotonin is one of the representative biogenic amines produced from tryptophan in the gastrointestinal tract as well as in the brain. The pineal gland also synthesizes serotonin, but as an intermediate in the biosynthesis of melatonin. The first enzymic step in the biosynthesis of serotonin is the hydroxylation of L-tryptophan catalyzed by tryptophan 5-monooxygenase. The 5-hydroxy-L-tryptophan (L-5HTP) formed is then decarboxylated to serotonin. Since the second step is catalyzed by non-specific aromatic L-amino acid decarboxylase, whether or not a given cell has the capacity to synthesize serotonin depends on the existence of tryptophan monooxygenase, and regulation of the serotonin biosynthesis is achieved mainly by modulating the activity of the first step enzyme. The activity of tryptophan monooxygenase was detected in extracts of the mouse stomach and intestines, although this activity was as low as approximately 1/20 of that in similar extract of the mouse brainstem. The upper small intestine and colon had a higher level of activity than other parts, and in the upper small intestine the enzyme was found to reside primarily in enterochromaffin cells of the mucosa. The intestinal tryptophan monooxygenase shared a common antigenic character with the enzyme from murine mastocytoma and was immunologically different from the brain enzyme. The enzymic properties were also similar to those of mastocytoma tryptophan monooxygenase which requires loosely-bound functional iron (Fe2+) for the activity. It seemed likely that the activity of tryptophan monooxygenase in paraneurons such as mastocytoma and enterochromaffin cells depends on available ferrous iron, and is therefore regulated, at least in part, by the cytosolic level of chelatable iron.

Detection of the IAA Biosynthetic Pathway from Tryptophan via Indole-3-Acetamide in <italic>Bradyrhizobium</italic> spp.

The IAA biosynthetic pathway of tryptophan to IAA via IAM was detected inBradyrhizobium spp. (slow-growing Rhizobium) but not in Rhizobium spp. (fast-growingRhizobium). A simple method using rapid HPLC analysis to measure the conversion from NAMto NAA was developed to detect indole-3-acetamide hydrolase activity in cultures of bacteria.Most of the Bradyrhizobium strains produce large amounts of NAA converted from NAM underour assay conditions. In addition, GC/MS analysis of purified extracts from cultures ofB.japonicum wild-type strain J1063, grown in a tryptophan-supplemented liquid medium, dem-onstrated the presence of IAM and IAA. The results strongly suggest that biosynthesis of IAA inBradyrhizobium spp. involves the same pathway as that operating in Pseudomonas savastanoiand Agrobacterium tumefaciens.Key words: Bradyrhizobium — IAA Biosynthesis — Indole-3-acetamide hydrolase.Microbial production of plant homones seems to beessential for the virulence of bacteria in their host plants(Morris 1986). For example, two well-studied phytopatho-genic bacteria, Pseudomonas syringae pv. savastanoi andAgrobacterium tumefaciens, possess a set of plant hor-mone biosynthetic genes. Gall formation on olive andoleander caused by the former bacterium, P. savastanoi,has been shown to be dependent on the bacterial synthesisof the plant hormone IAA (Comai and Kosuge 1980,1982). P. savastanoi produces IAA by means of tryp-tophan 2-monooxygenase and indole-3-acetamide hydro-lase according to the following pathway —• : tryptophanIAM -*• IAA (Fig. 1). Mutants deficient in genes of thispathway exhibit attenuated virulence. The latter bacte-rium, A. tumefaciens, causes crown gall disease in a widerange of plants (Nester et al. 1984). The formation ofcrown gall is associated with the presence of a portion ofthe Ti-plasmid, the T-DNA, which becomes an integratedAbbreviations: IAM, indole-3-acetamide; NAM, a-naph-thaleneacetamide; NAA, a-naphthaleneacetic acid; IPyA, indole-3-pyruvic acid; IEt, indole-3-ethanol; HPLC, high performanceliquid chromatography, GC/MS; gas chromatography/mass spec-trometry; ODS, octadesyl silanized silica gel; THF, tetrahydro-furan; t

The effects of overproduction of two Agrobacterium tumefaciens T-DNA auxin biosynthetic gene products in transgenic petunia plants

Two genes of Agrobacterium tumefaciens encode enzymes that together produce indoleacetic acid (IAA). The first gene, iaaM, encodes tryptophan monooxygenase which converts tryptophan to indoleacetamide (IAM). The second gene, iaaH, encodes indoleacetamide hydrolase which converts IAM to IAA. We have engineered each of the two genes to be expressed at either high constitutive levels or in a tissue-specific manner. These chimeric genes were introduced separately into petunia plants. The transgenic plants with the Agrobacterium iaaH gene are morphologically normal but have gained the ability to use IAM as an auxin. The plants with iaaM, in contrast, are morphologically abnormal. The observed abnormalities are consistent with an overproduction of auxin and ethylene. These plants contain an approximately 10-fold excess of IAA. The consequences of this deliberate manipulation of the normal phytohormone balance in transgenic plants are described.

Agrobacterium T-DNA gene 1 codes for tryptophan 2-monooxygenase activity in tobacco crown gall cells

Cloned tobacco crown gall tissue induced by the Agrobacterium tumefaciens C58 T-DNA mutant pGV3132, defective for the T-DNA-encoded amihydrolase ( iaaH), accumulates about 1000-times more indole-3-acet-amide (IAM) when compared to untransformed tobacco callus and crown gall tissue induced by a T-DNA mutant defective for gene 1. In vitro experiments demonstrated that this IAM accumulation is correlated with the active conversion of Trp into IAM. The results presented in this report provide biochemical evidence that the T-DNA gene 1 encodes a tryptophan 2-monooxygenase ( iaaM) activity in transformed plant cells. This gene cooperates with the gene 2-encoded amidohydrolase ( iaaH) in the T-DNA-controlled indole-3-acetic acid (IAA) biosynthesis pathway in crown gall cells.

Nucleotide sequences of the Pseudomonas savastanoi indoleacetic acid genes show homology with Agrobacterium tumefaciens T-DNA

We report the nucleotide sequences of iaaM and iaaH, the genetic determinants for, respectively, tryptophan 2-monooxygenase and indoleacetamide hydrolase, the enzymes that catalyze the conversion of L-tryptophan to indoleacetic acid in the tumor-forming bacterium Pseudomonas syringae pv. savastanoi. The sequence analysis indicates that the iaaM locus contains an open reading frame encoding 557 amino acids that would comprise a protein with a molecular weight of 61,783; the iaaH locus contains an open reading frame of 455 amino acids that would comprise a protein with a molecular weight of 48,515. Significant amino acid sequence homology was found between the predicted sequence of the tryptophan monooxygenase of P. savastanoi and the deduced product of the T-DNA tms-1 gene of the octopine-type plasmid pTiA6NC from Agrobacterium tumefaciens. Strong homology was found in the 25 amino acid sequence in the putative FAD-binding region of tryptophan monooxygenase. Homology was also found in the amino acid sequences representing the central regions of the putative products of iaaH and tms-2 T-DNA. The results suggest a strong similarity in the pathways for indoleacetic acid synthesis encoded by genes in P. savastanoi and in A. tumefaciens T-DNA.

Regulation of 3-indoleacetic acid production in Pseudomonas syringae pv. savastanoi. Purification and properties of tryptophan 2-monooxygenase.

The oxidative decarboxylation of L-tryptophan to yield 3-indoleacetamide, catalyzed by tryptophan 2-monooxygenase, represents a controlling reaction in the synthesis of indoleacetic acid by Pseudomonas savastanoi (Pseudomonas syringae pv. savastanoi), a gall-forming pathogen of olive (Olea europea L.) and oleander (Nerium oleander L.). Production of indoleacetic acid is essential for virulence of the bacterium in its hosts. Tryptophan 2-monooxygenase was characterized to determine its role in indoleacetic acid metabolism in the bacterium. The enzyme was purified to apparent homogeneity from Escherichia coli cells containing the genetic locus for this enzyme obtained from P. savastanoi. The preparation contained a single polypeptide with a mass of 62,000 that cross-reacted immunologically with a homologous protein in P. savastanoi. The holoenzyme contained one FAD moiety/subunit with properties consistent with a catalytic function. The enzyme preparation catalyzed an L-tryptophan-dependent O2 uptake and yielded 3-indoleacetamide as a product. Enzyme activity fit simple Michaelis Menten kinetics with a Km for L-tryptophan of 50 microM. 3-Indoleacetamide and 3-indoleacetic acid were identified as regulatory effectors. The apparent Ki for 3-indoleacetamide was 7 microM; that for indoleacetic acid was 225 microM. At Km concentrations of tryptophan, enzyme activity was inhibited 50% by 25 microM 3-indoleacetamide. In contrast, 230 microM indoleacetic acid was required to effect a similar inhibition. Phenylalanine and tyrosine were ineffective as regulatory metabolites. These results indicate that IAA synthesis in P. savastanoi is regulated by limiting tryptophan and by feedback inhibition from indoleacetamide and indoleacetic acid.

Cloning characterization of iaaM, a virulence determinant of Pseudomonas savastanoi.

Genes for indoleacetic acid production (iaaM and iaaH) are necessary for gall induction by the olive pathogen Pseudomonas savastanoi. In strain 2009 these determinants are borne on plasmid pIAA1. To map and characterize the genes, fragments of pIAA1 generated by EcoRI endonuclease treatment were cloned in Escherichia coli by using plasmid RSF1010 as vector. We isolated a recombinant plasmid encoding iaaM, the locus for tryptophan 2-monooxygenase. This plasmid, called pLUC1, was characterized by restriction endonuclease hydrolysis. It contained a 2.75-kilobase-pair segment of pIAA1. By cloning this segment in the EcoRI site of pBR328 and pBRH3B we showed that efficient expression of iaaM was dependent on the orientation with respect to the vector promoters, and thus determined the direction of transcription. To more finely map iaaM and confirm the orientation of transcription, plasmid pLUC1 was subjected to transposon Tn/mutagenesis. The promoter-distal end of iaaM was mapped between coordinates at 1.7 and 2.15 kilobase pairs of the cloned segment.

Involvement of plasmid deoxyribonucleic acid in indoleacetic acid synthesis in Pseudomonas savastanoi.

Olive (or oleander) knot is a plant disease incited by Pseudomonas savastanoi. Disease symptoms consist of tumorous outgrowths induced in the plant by bacterial production of indole-3-acetic acid (IAA). Synthesis of IAA occurs by the following reactions: L-tryptophan leads to indoleacetamide leads to indoleacetic acid, catalyzed by tryptophan 2-monooxygenase and indoleacetamide hydrolase, respectively. Whereas the enzymology of IAA synthesis is well characterized, nothing is known about the genetics of the system. We devised a positive selection for the presence of tryptophan 2-monooxygenase based on its capacity to use as a substrate the toxic tryptophan analogue 5-methyltryptophan. Efficient curing of the bacterium of tryptophan 2-monoxygenase, indoleacetamide hydrolase, and IAA production was obtained by acridine orange treatment. Further, loss of capacity to produce IAA by curing was correlated with loss of a plasmid of 34 X 10(6) molecular weight. This plasmid, here called pIAA1, when reintroduced into Iaa- mutants by transformation, restored tryptophan 2-monooxygenase and indoleacetamide hydrolase activities and production of IAA.

TRYPTOPHAN LOADING: CONSEQUENT EFFECTS ON THE SYNTHESIS OF KYNURENINE AND 5‐HYDROXYINDOLES IN RAT BRAIN

Abstract— Tryptophan loading of rats resulted in a continuous non‐linear uptake of l‐tryptophan from plasma into the brain. The optimum tryptophan load for increasing cerebral 5‐hydroxytryptamine (5‐HT) level was 25 mg/kg. Above this, there was a gradual decrease both in the levels and synthesis of 5‐HT and 5‐hydroxyindoleacetic acid (5‐HIAA) as assessed from simultaneous intraperitoneal or intraventricular injections of l[14C]tryptophan. A 5–10 fold increase in cerebral tryptophan produced a limited stimulation of 5‐HT synthesis. When the cerebral tryptophan level reached 1 ± 10‐4, substrate inhibition in vivo of the tryptophan monooxygenase (tryptophan‐5‐hydroxylase) but not of the indoleamine‐2,3‐dioxygenase occurred. Cerebral synthesis of kynurenine increased linearly with increasing tryptophan load. At a plasma ratio of 50:1 tryptophan to kynurenine, tryptophan loading interfered with the entry of peripheral kynurenine. Tryptophan loading also increased the efflux of 5‐hydroxyindoles from the brain. One hour after intraperitoneal injection of l‐kynurenine sulfate (5 mg/kg) into rats, there was a shift in the plasma ratio of l‐tryptophan to l‐kynurenine to 4:1. In these rats, a 20% reduction of cerebral tryptophan was noted.