Structures of Grixazone A and B, A-Factor-dependent Yellow Pigments Produced under Phosphate Depletion by Streptomyces griseus

Ohnishi, Yasuo; Furusho, Yasuhide; Higashi, Tatsuichiro; Chun, Hyo Kon; Furihata, Kazuo; Sakuda, Shohei; Horinouchi, Sueharu

doi:10.7164/antibiotics.57.218

Cited by 33 publications

(27 citation statements)

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“…1A), a mixture of yellow pigments grixazone A and grixazone B, containing a phenoxazinone chromophore, as secondary metabolites of Streptomyces griseus (9,10). In the present study on the grixazone biosynthesis, we found that 3-amino-4-hydroxybenzoic acid (3,4-AHBA, Fig.…”

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confidence: 69%

Novel Benzene Ring Biosynthesis from C3 and C4 Primary Metabolites by Two Enzymes

Suzuki

Ohnishi

Furusho

et al. 2006

Journal of Biological Chemistry

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The shikimate pathway, including seven enzymatic steps for production of chorismate via shikimate from phosphoenolpyruvate and erythrose-4-phosphate, is common in various organisms for the biosynthesis of not only aromatic amino acids but also most biogenic benzene derivatives. 3-Amino-4-hydroxybenzoic acid (3,4-AHBA) is a benzene derivative serving as a precursor for several secondary metabolites produced by Streptomyces, including grixazone produced by Streptomyces griseus. Our study on the biosynthesis pathway of grixazone led to identification of the biosynthesis pathway of 3,4-AHBA from two primary metabolites. Two genes, griI and griH, within the grixazone biosynthesis gene cluster were found to be responsible for the biosynthesis of 3,4-AHBA; the two genes conferred the in vivo production of 3,4-AHBA even on Escherichia coli. In vitro analysis showed that GriI catalyzed aldol condensation between two primary metabolites, L-aspartate-4-semialdehyde and dihydroxyacetone phosphate, to form a 7-carbon product, 2-amino-4,5-dihydroxy-6-one-heptanoic acid-7-phosphate, which was subsequently converted to 3,4-AHBA by GriH. The latter reaction required Mn 2؉ ion but not any cofactors involved in reduction or oxidation. This pathway is independent of the shikimate pathway, representing a novel, simple enzyme system responsible for the synthesis of a benzene ring from the C 3 and C 4 primary metabolites.The shikimate pathway (Fig. 1B), involving seven enzymatic steps that produce chorismate via shikimate from phosphoenolpyruvate (PEP) 2 and erythrose-4-phosphate, is well established as the common pathway for the biosynthesis of aromatic amino acids in bacteria, fungi, algae, and higher plants. Not only aromatic amino acids but also most biogenic benzene derivatives, such as p-aminobenzoic acid, m-aminobenzoic acid, 2-amino-3-hydroxybenzoic acid, 2-amino-6-hydroxybenzoic acid, and many vitamins, are derived from chorismate (1, 2). The shikimate biosynthesis pathway is also employed by Archaea, although the genes encoding the first two enzymes involved in 3-dehydroquinate (DHQ) synthesis are missing in the genomic sequences of many Archaea (3). In one of Archaea, Methanocaldococcus jannaschii, DHQ is synthesized from aspartate 4-semialdehyde (ASA) and 6-deoxy-5-ketofructose-1-phosphate by two alternative enzymes and supplied to the shikimate pathway (4). Recent studies (5, 6) showed that 3-amino-5-hydroxybenzoic acid, a precursor for ansamycin antibiotics, is also synthesized through the aminoshikimate pathway, a variant of the shikimate pathway. Thus, the benzene ring as one of the primary chemical structures in nature is extensively formed through the shikimate pathway, although some benzene derivatives are formed from aliphatic acyl-CoA by polyketide synthases (7, 8).We recently isolated grixazone (Fig. 1A), a mixture of yellow pigments grixazone A and grixazone B, containing a phenoxazinone chromophore, as secondary metabolites of Streptomyces griseus (9, 10). In the present study on the grixazone biosynthesis, we ...

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confidence: 69%

Novel Benzene Ring Biosynthesis from C3 and C4 Primary Metabolites by Two Enzymes

Suzuki

Ohnishi

Furusho

et al. 2006

Journal of Biological Chemistry

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“…Identification of 3,4-AHBAL and 3-Acetylamino-4-hydroxybenzaldehyde (3, in the Supernatant of S. griseus Mutant ⌬griEF-For the purpose of overproduction of grixazone and its intermediates, we placed griR under the control of the melC promoter in pIJ702. The griR-coding sequence was amplified by the following two primers, 5Ј-GCGCATGCCTACGCACCATC-3Ј (with the start codon of griR in italic letters and the SphI site underlined) and 5Ј-TCGAATTCTCAGC-CGGAGCGCCC-3 (with the stop codon of griR in italic letters and the EcoRI site underlined).…”

Section: Methodsmentioning

confidence: 99%

“…In this study, we determined the plausible substrate of GriF by structural elucidation of an intermediate (3-amino-4-hydroxybenzaldehyde (3, ; compound 2a) (see Fig. 2C) that accumulated in a griEFdeleted mutant and revealed the in vitro formation of a phenoxazinone skeleton from 3,4-AHBAL by recombinant GriF produced in Escherichia coli.…”

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A Novel o-Aminophenol Oxidase Responsible for Formation of the Phenoxazinone Chromophore of Grixazone

Suzuki

Furusho

Higashi

et al. 2006

Journal of Biological Chemistry

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Grixazone contains a phenoxazinone chromophore and is a secondary metabolite produced by Streptomyces griseus. In the grixazone biosynthesis gene cluster, griF (encoding a tyrosinase homolog) and griE (encoding a protein similar to copper chaperons for tyrosinases) are encoded. An expression study of GriE and GriF in Escherichia coli showed that GriE activated GriF by transferring copper ions to GriF, as has been observed for a Streptomyces melanogenesis system in which the MelC1 copper chaperon transfers copper ions to MelC2 tyrosinase. In contrast with tyrosinases, GriF showed no monophenolase activity, although it oxidized various o-aminophenols as preferable substrates rather than catechol-type substrates. Deletion of the griEF locus on the chromosome resulted in accumulation of 3-amino-4-hydroxybenzaldehyde (3,4-AHBAL) and its acetylated compound, 3-acetylamino-4-hydroxybenzaldehyde. GriF oxidized 3,4-AHBAL to yield an o-quinone imine derivative, which was then non-enzymatically coupled with another molecule of the o-quinone imine to form a phenoxazinone. The coexistence of N-acetylcysteine in the in vitro oxidation of 3,4-AH-BAL by GriF resulted in the formation of grixazone A, suggesting that the -SH group of N-acetylcysteine is conjugated to the o-quinone imine formed from 3,4-AHBAL and that the conjugate is presumably coupled with another molecule of the o-quinone imine. GriF is thus a novel o-aminophenol oxidase that is responsible for the formation of the phenoxazinone chromophore in the grixazone biosynthetic pathway.We have long studied the A-factor regulatory cascade that leads to secondary metabolite formation and morphological differentiation in Streptomyces griseus (1, 2). The A-factor (2-isocapryloyl-3R-hydroxymethyl-␥-butyrolactone) triggers the synthesis of almost all the secondary metabolites produced by this species. One of the secondary metabolites under the control of the A-factor is grixazone. Grixazone is a yellow pigment and actually a mixture of grixazones A and B (compounds 1a and 1b) (see Fig. 2C) (3). Grixazone A is a novel compound, and grixazone B has been reported to show a parasiticide activity (4).Grixazones contain a phenoxazinone chromophore. The phenoxazinone skeleton is common to actinomycin D produced by Streptomyces antibioticus (5), michigazone produced by Streptomyces michiganensis (6), texazone produced by Streptomyces sp. WRAT-210 (7), exfoliazone produced by Streptomyces exfoliatus (8), and 4-demethoxymichigazone produced by Streptomyces halstedii (9). Hsieh and Jones (10) reported a phenoxazinone synthase in S. antibioticus that catalyzes the six-electron oxidative coupling of o-aminophenol compounds derived from tryptophan through 3-hydroxyanthranilic acid. However, disruption of the phenoxazinone synthase gene in S. antibioticus does not affect actinomycin D synthesis, showing that the phenoxazinone skeleton in actinomycin D is biosynthesized in vivo by a still unknown enzyme or non-enzymatically (11). On the other hand, michigazone with a hydroxymethyl group at ...

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“…WRAT-210. [12][13][14][15][16] Phenoxazinones frequently encountered in S. griseus strains include the grixazones and the elloxazinones, [11,17] all of which lack C-9 functionalization. Whereas the grixazones bear an N-acetylated cysteine moiety at C-1 through a thioether bond, the elloxazinones possess a carboxamide function in this position.…”

Section: Resultsmentioning

confidence: 99%

Bezerramycins A–C, Antiproliferative Phenoxazinones from Streptomyces griseus Featuring Carboxy, Carboxamide or Nitrile Substituents

et al. 2009

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Three new phenoxazinone antibiotics, named bezerramycins A–C (1–3), were isolated from a Streptomyces griseus strain (HKI 0545, DSM 41823) together with the known metabolite elloxazinone B (4). Their structures were elucidated by one‐ and two‐dimensional NMR techniques (1H, 13C, HSQC, 1H‐1H COSY and HMBC) as well as mass spectrometry. One of the isolated phenoxazinones (bezerramycin C) features a nitrile moiety, which is scarce in natural products. All compounds were evaluated for their antiproliferative and cytotoxic activities. Those featuring a hydroxymethyl substituent at C‐8 were found to exhibit moderate antiproliferative properties against vascular endothelium cells (HUVEC) (GI50 = 14.5–20 μM). A biosynthetic model for the isolated compounds is discussed.

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Structures of Grixazone A and B, A-Factor-dependent Yellow Pigments Produced under Phosphate Depletion by Streptomyces griseus

Cited by 33 publications

References 9 publications

Novel Benzene Ring Biosynthesis from C3 and C4 Primary Metabolites by Two Enzymes

Novel Benzene Ring Biosynthesis from C3 and C4 Primary Metabolites by Two Enzymes

A Novel o-Aminophenol Oxidase Responsible for Formation of the Phenoxazinone Chromophore of Grixazone

Bezerramycins A–C, Antiproliferative Phenoxazinones from Streptomyces griseus Featuring Carboxy, Carboxamide or Nitrile Substituents

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