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 ...

Grixazone (GX), which is a diffusible yellow pigment containing a phenoxazinone chromophore, is one of the secondary metabolites under the control of A-factor (2-isocapryloyl-3R-hydroxymethyl-␥-butyrolactone) in Streptomyces griseus. GX production is also induced by phosphate starvation. The whole biosynthesis gene cluster for GX was cloned and characterized. The gene cluster consisting of 13 genes contained six transcriptional units, griT, griSR, griR, griAB, griCDEFG, and griJIH. During cultivation in a phosphate-depleted medium, the six promoters were activated in the order (i) griR, (ii) griC and griJ, and (iii) griT, griS, and griA. Disruption of griR, which encodes a SARP family transcriptional regulator, abolished the transcriptional activation of all other genes in the cluster. In addition, ectopic expression of griR from a constitutively active promoter resulted in GX overproduction even in the absence of AdpA, a key transcriptional activator in the A-factor regulatory cascade, and in the presence of phosphate at a high concentration. GriR monomers bound direct repeat sequences in the griC and griJ promoters in a cooperative manner. Therefore, the early active genes (griCDEFG and griJIH), all of which, except for griG (which encodes a transporter-like protein), encode the GX biosynthesis enzymes, were directly activated by GriR. The transcription of griR was greatly reduced in the presence of phosphate at a high concentration and was hardly detected in the absence of AdpA. These findings showed that both A-factor and phosphate depletion signals were required for griR transcription and both signals were transmitted to the GX biosynthesis genes solely via the griR promoter.A-factor (2-isocapryloyl-3R-hydroxymethyl-␥-butyrolactone; see Fig. 7 for its structure) is a chemical signaling molecule, or a microbial hormone, that triggers secondary metabolism and cell differentiation in Streptomyces griseus (10,25). A-factor is gradually accumulated in a growthdependent manner by the activity of AfsA, which is the key enzyme for A-factor biosynthesis. We have recently established the whole A-factor biosynthesis pathway, including the function of AfsA, which catalyzes ␤-ketoacyl transfer between dihydroxyacetone phosphate and 8-methyl-3-oxononanoyl-acyl carrier protein (12). When the concentration of A-factor reaches a critical level at or near the middle of the exponential growth phase, it binds the A-factor receptor protein (ArpA), which has bound and repressed the promoter of adpA, and dissociates ArpA from the promoter, thus inducing transcription of adpA (26). AdpA then activates a number of genes required for secondary metabolism and morphological differentiation, forming an AdpA regulon (13, 27). Members of the AdpA regulon that are involved in secondary metabolism include strR, the pathway-specific transcriptional activator for streptomycin biosynthesis (38), and an open reading frame (ORF) that encodes a probable pathwayspecific regulator for a polyketide compound (39).Grixazone (GX) is a diffusible yello...

that induces morphological development and secondary metabolism in Streptomyces griseus. A diffusible yellow pigment is produced by S. griseus in an A-factor-dependent manner under phosphate depletion. Detailed analysis of the pigment production by S. griseus cultivated in minimal liquid medium containing different concentrations of phosphate showed that the pigment was actively produced in the presence of low concentrations of phosphate and the production of the pigment was completely repressed in the presence of 2.5mM KH2PO4. HPLC analysis of the culture supernatant showed that the pigment consisted of two major, structurally related compounds and they were produced at different ratios depending on the concentration of phosphate in the medium. The structures of the two major compounds, designated as grixazone A and B, were determined by spectroscopic analyses as 1-[[2-

Compound (Ib) is reported to be a known parasiticide. -(OHNISHI, Y.; FURUSHO, Y.; HIGASHI, T.; CHUN, H.-K.; FURIHATA, K.; SAKUDA, S.; HORINOUCHI*, S.; J. Antibiot. 57 (2004) 3, 218-223; Dep. Appl. Biol. Chem., Grad. Sch. Agric. Life Sci., Univ. Tokyo, Bunkyo, Tokyo 113, Japan; Eng.) -C. Oppel 36-174