Biosynthesis of Indolocarbazole and Goadsporin,Two Different Heterocyclic Antibiotics Produced by Actinomycetes

The biosynthesis of staurosporine, rebeccamycin, and goadsporin, which are produced by actinomycetes and contain characteristic heterocyclic rings, was characterized by genetic methods. Staurosporine and rebeccamycin contain an indolocarbazole ring synthesized from two molecules of tryptophan, with indolepyruvic acid imine and chromopyrrolic acid as biosynthetic intermediates. A tetrameric hemoprotein synthesizes chromopyrrolic acid, and cytochrome P450 peroxidase catalyzes the intramolecular C–C coupling and decarboxylation of chromopyrrolic acid to form the indolocarbazole core. Goadsporin is a thiopeptide containing thiazole and oxazole heterocyclic rings. The structural gene godA is ribosomally translated to a goadsporin precursor peptide, and oxazole, methyloxazole, and thiazole rings are derived from serine, threonine, and cystein through post-translational modifications. On the basis of these knowledges, a wide variety of indolocarbazole and goadsporin analogs through the rational gene recombination and disruption of these biosynthetic genes were successfully produced.     

Crystal structure of VioE, a key player in the construction of the molecular skeleton of violacein

Violacein and the indolocarbazoles are naturally occurring bisindole products with various biological activities, including antitumor activity. Although these compounds have markedly different molecular skeletons, their biosynthetic pathways share the same intermediate "compound X," which is produced from L-tryptophan via indole-3-pyruvic acid imine. Compound X is a short-lived intermediate that is spontaneously converted to chromopyrrolic acid for indolocarbazole biosynthesis, whereas VioE transforms compound X into protodeoxyviolaceinic acid, which is further modified by other enzymes to produce violacein. Thus, VioE plays a key role in the construction of the molecular skeleton of violacein. Here, we present the crystal structure of VioE, which consists of two subunits, each of which forms a structure resembling a baseball glove. Each subunit has a positively charged pocket at the center of the concave surface of the structure. Mutagenesis analysis of the surface pocket and other surface residues showed that the surface pocket serves as an active site. We have also solved the crystal structure of a complex of VioE and phenylpyruvic acid as an analogue of a VioE-substrate complex. A docking simulation with VioE and the IPA imine dimer, which is proposed to be compound X, agreed with the results from the mutational analysis and the VioE-phenylpyruvic acid complex structure. Based on these results, we propose that VioE traps the highly reactive substrate within the surface pocket to suppress CPA formation and promote protodeoxyviolaceinic acid formation caused by proximity and orientation effects.     

Molecular analysis of the rebeccamycin L-amino acid oxidase from Lechevalieria aerocolonigenes ATCC 39243

Rebeccamycin, a member of the tryptophan-derived indolocarbazole family, is produced by Lechevalieria aerocolonigenes ATCC 39243. The biosynthetic pathway that specifies biosynthesis of this important metabolite is comprised of 11 genes spanning 18 kb of DNA. A presumed early enzyme involved in elaboration of the rebeccamycin aglycone is encoded by rebO, located at the left-hand region of the reb gene cluster. The deduced protein product, RebO (51.9 kDa), is an L-amino acid oxidase (L-AAO) that has 27% identity to an L-AAO from Scomber japonicus (animal, mackerel) and is a member of the family of FAD-dependent oxidase enzymes. In order to study the biochemical properties of this key enzyme, the rebO gene was overexpressed and purified from Escherichia coli. Biochemical characterization showed that RebO is dimeric, with a molecular mass of approximately 101 kDa. Further analysis revealed that the enzyme contains a noncovalently bound FAD cofactor and is reoxidized at the expense of molecular oxygen by producing one molecule of hydrogen peroxide. Based on kinetic studies, RebO shows significant preference for 7-chloro-L-tryptophan, suggesting its likely role as the natural early pathway substrate. Furthermore, the native RebO enzyme has evident, albeit limited, flexibility as shown by bioconversion studies with unnatural substrates. This work provides the first analysis of a structural enzyme involved in construction of this important class of indolocarbazole natural products.     

Gibberellin-enhanced indole-3-acetic acid biosynthesis: D-Tryptophan as the precursor of indole-3-acetic acid

Stem segments excised from light-grown Pisum sativum L. (cv. Little Marvel) plants elongated in the presence of indole-3-acetic acid and its precursors, except for L-tryptophan, which required the addition of gibberellin A, for induction of growth. Segment elongation was promoted by D-tryptophan without a requirement for gibberellin, and growth in the presence of both D-tryptophan and L-tryptophan with gibberellin A3, was inhibited by the D-aminotransferase inhibitor D-cycloserine. Tryp-tophan racemase activity was detected in apices and promoted conversion of L-tryptophan to the D isomer; this activity was enhanced by gibberellin A3. When applied to apices of intact untreated plants, radiolabeled D-tryptophan was converted to indole-3-acetic acid and indoleacetylaspartic acid much more readily than L-tryptophan. Treatment of plants with gibberellin A3, 3 days prior to application of labeled tryptophan increased conversion of L-tryptophan to the free auxin and its conjugate by more than 3-fold, and led to labeling of N-malonyl-D-tryptophan. It is proposed that gibberellin increases the biosynthesis of indole-3-acetic acid by regulating the conversion of L-tryptophan to D-tryptophan, which is then converted to the auxin.     

Tryptophol Formation by Zygosaccharomyces priorianus

Zygosaccharomyces priorianus converted L-tryptophan to tryptophol and to small quantities of indole-3-acetic acid. Neither tryptophol nor indole-3-acetic acid was metabolized when added separately to growing cultures. The possible intermediacy of indole-3-pyruvic acid, indole-3-acetaldehyde, and tryptamine in the degradation of L-tryptophan was tested by feeding these compounds to Z. priorianus and Saccharomyces cerevisiae. Indole-3-pyruvic acid and indole-3-acetaldehyde were converted to tryptophol and indole-3-acetic acid, with the latter accumulating only in small amounts. Tryptamine was converted to its N-acetyl derivative by these organisms. A qualitative study was made on the metabolism of L-phenylalanine, L-tyrosine, and L-5-hydroxytryptophan by these organisms. Like L-tryptophan, these amino acids were metabolized to their respective alcohol and acid derivatives. Of a large number of organisms tested, the yeasts possessed the highest capacity for degrading L-tryptophan to tryptophol.     

Production of indole-3-acetic acid and related indole derivatives from L-tryptophan by <Emphasis Type="Italic">Rubrivivax benzoatilyticus</Emphasis> JA2

Rubrivivax benzoatilyticus JA2 produces indoles with simultaneous utilization of L-tryptophan. Fifteen chromatographically distinct indole derivatives were detected from the L-tryptophan-supplemented cultures of R. benzoatilyticus JA2. Nine of these were identified as, indole 3-acetamide, Methoxyindole-3-aldehyde, indole 3-aldehyde, methoxyindole-3-acetic acid, indole 3-acetic acid, indole-3-carboxylic acid, indole-3-acetonitrile, indole, and trisindoline. Tryptophan stable isotope feeding confirmed the indoles produced are from the supplemented L-tryptophan. Indole 3-acetic acid is one of the major products of L-tryptophan catabolism by R. benzoatilyticus JA2 and its production was influenced by growth conditions. Identification of indole 3-acetamide and tryptophan monooxygenase activity suggests indole 3-acetamide routed IAA biosynthesis in R. benzoatilyticus JA2. The study also indicated the possible multiple pathways of IAA biosynthesis in R. benzoatilyticus JA2.     

Purification and characterization of indolepyruvate decarboxylase. A novel enzyme for indole-3-acetic acid biosynthesis in Enterobacter cloacae.

Indolepyruvate decarboxylase, a key enzyme for indole-3-acetic acid biosynthesis, was found in extracts of Enterobacter cloacae. The enzyme catalyzes the decarboxylation of indole-3-pyruvic acid to yield indole-3-acetaldehyde and carbon dioxide. The enzyme was purified to apparent homogeneity from Escherichia coli cells harboring the genetic locus for this enzyme obtained from E. cloacae. The results of gel filtration experiments showed that indolepyruvate decarboxylase is a tetramer with an M(r) of 240,000. In the absence of thiamine pyrophosphate and Mg2+, the active tetramers dissociate into inactive monomers and dimers. However, the addition of thiamine pyrophosphate and Mg2+ to the inactive monomers and dimers results in the formation of active tetramers. These results indicate that the thiamine pyrophosphate-Mg2+ complex functions in the formation of the tetramer, which is the enzymatically active holoenzyme. The enzyme exhibited decarboxylase activity with indole-3-pyruvic acid and pyruvic acid as substrates, but no decarboxylase activity was apparent with L-tryptophan, indole-3-lactic acid, beta-phenylpyruvic acid, oxalic acid, oxaloacetic acid, and acetoacetic acid. The Km values for indole-3-pyruvic acid and pyruvic acid were 15 microM and 2.5 mM, respectively. These results indicate that indole-3-acetic acid biosynthesis in E. cloacae is mediated by indolepyruvate decarboxylase, which has a high specificity and affinity for indole-3-pyruvic acid.     

利用大肠杆菌细胞工厂生产吲哚-3-乙酸的研究

目的:利用重组大肠杆菌全细胞转化色氨酸生产IAA.方法:在大肠杆菌胞内构建两条全新的IAA合成途径,即吲哚-3-乙酰胺(indole-3-acetamide,IAM)途径和色胺(tryptamine,TRP)途径.结果:IAM途径涉及两个酶,分别是色氨酸-2-单加氧酶(IAAM)和酰胺酶(AMI1),构建好的重组大肠杆...     

Stereochemistry and mechanism of a new single-turnover, half-transamination reaction catalyzed by the tryptophan synthase alpha 2 beta 2 complex

Tryptophan synthase is a versatile enzyme that catalyzes a wide variety of pyridoxal phosphate dependent reactions that are also catalyzed in model systems. These include beta-replacement, beta-elimination, racemization, and transamination reactions. We now show that the apo-alpha 2 beta 2 complex of tryptophan synthase will bind two unnatural substrates, pyridoxamine phosphate and indole-3-pyruvic acid, and will convert them by a single-turnover, half-transamination reaction to pyridoxal phosphate and L-tryptophan, the natural coenzyme and a natural product, respectively. This enzyme-catalyzed reaction is more rapid and more stereospecific than an analogous model reaction. The pro-S 4'-methylene proton of pyridoxamine phosphate is removed during the reaction, and the product is primarily L-tryptophan. We conclude that pyridoxal phosphate enzymes may be able to catalyze some unnatural reactions involving bound reactants and bound coenzyme since the coenzyme itself has the intrinsic ability to promote a variety of reactions.     

l-Tryptophan metabolism in wound-activated and Agrobacterium tumefaciens-transformed potato tuber cells

The in vivo metabolism of L-tryptophan in wound-activated and Agrobacterium tumefaciens , strain C 58, transformed tissues of white potato tubers ( Solanum tuberosum L. cv. Saskia) was investigated. The following metabolites of L-tryptophan were identified in both tissues by co-chromatography with authentic standards in several thinlayer chromotography (TLC) and high pressure liquid chromatographic (HPLC) systems: indole-3-acetic acid (IAA), indole-3-acetaldehyde, indole-3-ethanol, indole-3-acetamide and tryptamine. Labelled indole-3-acetaldoxime was only found in transformed tissue. Crown gall tissue generally incorporated [¹⁴C]-L-tryptophan into precursors of IAA at a distinctly higher rate than did wound tissue. Tryptamine and indole-3-ethanol accumulated about ten-fold more label in crown gall cells than in cells from wounded tissue. The incorporation of radioactivity into indole-3-acetamide as determined by 2 consecutive TLC systems followed by HPLC analysis was rather low, though consistently observed in both tissues. An indole-3-acetamide hydrolyzing enzyme, the putative product of gene 2 on the T-DNA, could be extracted from the transformed tissue only. The indole-3-ethanol level was 4.3 nmol (g dry weight)⁻¹ and 41 nmol (g dry weight)⁻¹ for wounded tissue and primary crown gall tissue, respectively, as determined by HPLC with a [¹⁴C]-labelled internal standard. The experiments are critically discussed in relation to recent reports on a T-DNA encoded enzyme of IAA biosynthesis in crown gall tumors.     

Localization of newly synthesized indoIe-3-methylglucosinolate (= glucobrassicin) in vacuoles from horseradish (Armoracia rusticana)

The presence and concentration of indole-3-methylglucosinolate [= glucobrassicin; 0.49 μmol (g dry weight)-1] and its 1-methoxy derivative [0.38 umol (g dry weight)⁻¹] in Armoracia rusticana P. Gärtner, B. Meyer and Scherb, storage roots were measured. The storage tissue contains L-tryptophan [1.63 μmol (g dry weight)^-1], which is the common precursor amino acid of the indoleglucosinolates. Tissue cylinders convert [¹⁴C]- l -tryptophan efficiently to the indoleglucosinolates (25%) in vivo. The conversion of [¹⁴C]- l -tryptophan to indole-3-methylgluco-sinolate shows biphasic kinetics.
A fraction rich in vacuoles was prepared from tissue sections to which [¹⁴C]- l -tryp-tophan had been fed and allowed to be metabolized. The predominantly vacuolar location of both L-tryptophan and the indoleglucosinolates is demonstrated by correlation with the vacuolar marker acid phosphatase.
The significance for the regulation of the indoleglucosinolate biosynthesis and the role of indole-3-methylglucosinolate as a potential auxin precursor are discussed.     

Presence of a Pathway for the Biosynthesis of Auxin via Indole-3-Acetamide in Trifoliata Orange

The auxin-biosynthetic pathway from L-tryptophan to indole-3-aceticadd via indole-3-acetamide (IAM), found in plant-pathogenicbacteria such as Agrobacterium tumefaciens and Pseudomonas savastanoi,has not been found in plants. We attempted to detect the enzymaticactivities for this pathway in cell-free systems from varioustissues of trifoliata orange (Poncirus trifoliata Rafin.). Ahigh level of activity of LAM hydrolase, which catalyzes theconversion of IAM to indole-3-acetic acid, was observed in acrude extract prepared from young fruits one week after fullbloom. Using -naphthaleneacetamide as a competitor of IAM hydrolase,a simple assay system was developed for the detection of theconversion of L-tryptophan to IAM (tryptophan monooxygenaseactivity). When this system was used to assay cell-free extractsof young fruit of P. trifoliata, the conversion of L-tryptophanto IAM was clearly demonstrated by the presence of IAM amongreaction products, as demonstrated by GC/MS analysis and theincorporation of ¹⁴C-labeled L-tryptophan into an IAM fraction.This is the first report indicating the presence of an auxin-biosyntheticpathway via IAM in P. trifoliata. Furthermore, it is shown thatboth enzyme activities in auxin biosynthesis increased transientlyduring fruit development. (Received October 9, 1992; Accepted November 2, 1992)     

The Excessive Production of Indole-3-Acetic Acid and Its Significance in Studies of the Biosynthesis of This Regulator of Plant Growth and Development

Because of the importance of indole-3-acetic acid (IAA) in thegrowth and development of plants, extensive studies of the biosynthesisof IAA have been performed during the four decades since thediscovery of IAA as a plant hormone. The pathway for the biosynthesisof IAA in plants remains, however, to be unelucidated, eventhough studies within the past decade have revealed unexpectedaspects of such biosynthesis. By contrast, two pathways to IAAhave been characterized in bacteria at the molecular level:the indole-3-acetamide (IAM) pathway (L-tryptophan     

Indole-3-lactic acid as a tryptophan metabolite produced by Bifidobacterium spp

Fifty-one strains of the genus Bifidobacterium have been found to accumulate indole-3-lactic acid in culture broth. The isolated metabolite was identified through mass and nuclear magnetic resonance spectroscopy. All the microorganisms tested, as resting cells, have been shown to be able to convert L-tryptophan into L-indole-3-lactic acid.     

Indole-3-lactic acid as a tryptophan metabolite produced by Bifidobacterium spp.

Fifty-one strains of the genus Bifidobacterium have been found to accumulate indole-3-lactic acid in culture broth. The isolated metabolite was identified through mass and nuclear magnetic resonance spectroscopy. All the microorganisms tested, as resting cells, have been shown to be able to convert L-tryptophan into L-indole-3-lactic acid.     

In vitro biosynthesis of violacein from L-tryptophan by the enzymes VioA-E from Chromobacterium violaceum

The purple chromobacterial pigment violacein arises by enzymatic oxidation and coupling of two molecules of l-tryptophan to give a rearranged pyrrolidone-containing scaffold in the final pigment. We have purified five contiguously encoded proteins VioA-E after expression in Escherichia coli and demonstrate the full 14-electron oxidation pathway to yield the final chromophore. The flavoenzyme VioA and the heme protein VioB work in conjunction to oxidize and dimerize l-tryptophan to a nascent product that can default to the off pathway metabolite chromopyrrolic acid. In the presence of VioE, the intermediate instead undergoes on-pathway [1,2] indole rearrangement to prodeoxyviolacein. The last two enzymes in the pathway are flavin-dependent oxygenases, VioC and VioD, that act sequentially. VioD hydroxylates one indole ring at the 5-position to yield proviolacein, and VioC then acts on the other indole ring at the 2-position to create the oxindole and complete violacein formation.     

Cloning and characterization of a locus encoding an indolepyruvate decarboxylase involved in indole-3-acetic acid synthesis in Erwinia herbicola.

Erwinia herbicola 299R synthesizes indole-3-acetic acid (IAA) primarily by the indole-3-pyruvic acid pathway. A gene involved in the biosynthesis of IAA was cloned from strain 299R. This gene (ipdC) conferred the synthesis of indole-3-acetaldehyde and tryptophol upon Escherichia coli DH5 alpha in cultures supplemented with L-tryptophan. The deduced amino acid sequence of the gene product has high similarity to that of the indolepyruvate decarboxylase of Enterobacter cloacae. Regions within pyruvate decarboxylases of various fungal and plant species also exhibited considerable homology to portions of this gene. This gene therefore presumably encodes an indolepyruvate decarboxylase (IpdC) which catalyzes the conversion of indole-3-pyruvic acid to indole-3-acetaldehyde. Insertions of Tn3-spice within ipdC abolished the ability of strain 299R to synthesize indole-3-acetaldehyde and tryptophol and reduced its IAA production in tryptophan-supplemented minimal medium by approximately 10-fold, thus providing genetic evidence for the role of the indolepyruvate pathway in IAA synthesis in this strain. An ipdC probe hybridized strongly with the genomic DNA of all E. herbicola strains tested in Southern hybridization studies, suggesting that the indolepyruvate pathway is common in this species. Maximum parsimony analysis revealed that the ipdC gene is highly conserved within this group and that strains of diverse geographic origin were very similar with respect to ipdC.