Phenoxazinone Biosynthesis: Accumulation of a Precursor, 4-Methyl-3-hydroxyanthranilic Acid, by Mutants of Streptomyces parvulus

1987

J Bacteriol

A methyltransferase which utilizes 3-hydroxyanthranilic acid (HAA) as a substrate was identified in detergent-treated extracts of the bacterium Streptomyces antibioticus. Much information regarding the enzymology of actinomycin biosynthesis has also been adduced. Phenoxazinone synthase (PHS) catalyzes the oxidative condensation of o-aminophenols and is thought to be involved in the synthesis of the actinomycin chromophore, actinocin, at least in Streptomyces antibioticus. The enzyme was first identified in S. antibioticus extracts by Katz and Weissbach (8) and has been purified to homogeneity in my laboratory (3). The gene for the 88,000-Mr PHS subunit has been cloned and used as a probe to study PHS regulation in S. antibioticus and in other streptomycetes (5-7). Keller et al. (11,12) have reported the characterization of an enzyme in Streptomyces chrysomallus which activates MHA for its subsequent participation in the polymerization reactions leading to the synthesis of MHA pentapeptides and of enzymes which modify and polymerize the amino acids in the pentapeptide chains.The present report describes the characterization of another enzyme which is apparently involved in the biosynthesis of actinomycin, a methyltransferase which catalyzes the conversion of 3-hydroxyanthranilic acid (HAA) to MHA. The reaction involved and a portion of the putative pathway for actinomycin biosynthesis are shown in Fig. 1.

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Radioisotopesmentioning

confidence: 99%

See 1 more Smart Citation

Actinomycin synthesis in Streptomyces antibioticus: enzymatic conversion of 3-hydroxyanthranilic acid to 4-methyl-3-hydroxyanthranilic acid

1987

J Bacteriol

“…A putative pathway for actinomycin biosynthesis was proposed several years ago (50), and biochemical, physiological, and genetic studies have confirmed the essential details of that pathway. Five enzymes from S. antibioticus, Streptomyces chrysomallus, and Streptomyces parvulus have been isolated and characterized to demonstrate their involvement in the actinomycin biosynthetic pathway (6,11,22,23,(28)(29)(30)(31).…”

mentioning

confidence: 99%

Nucleotide sequence, transcriptional analysis, and glucose regulation of the phenoxazinone synthase gene (phsA) from Streptomyces antibioticus

Hsieh

1995

J Bacteriol

The nucleotide sequence of a 2.3-kb SphI fragment containing the structural gene (phsA) for phenoxazinone synthase (PHS) of Streptomyces antibioticus was determined. The sequence was found to contain an open reading frame (ORF) with a G؉C content of 71.5% oriented in the direction of transcription that was confirmed by primer extension. The ORF encodes a protein with an M r of 70,223 consisting of 642 amino acids and is preceded by a potential ribosome-binding site. The codon usage pattern is in agreement with the general pattern for streptomycete genes, with a 92.5 mol% G؉C content in the third position. The N-terminal sequence of the mature PHS subunit corresponds exactly to that predicted from the nucleotide sequence. Neither ATG nor GTG initiator codons were identified for the protein. However, a TTG codon was located near the amino terminus of the mature protein and is a good candidate for the initiator codon. The transcriptional start point of phsA was located 36 bp upstream of the start codon by primer extension. The ؊10 region of the putative promoter showed some similarity to the consensus sequence for the major class of prokaryotic promoters, but the ؊35 region was less similar. Comparison of the primary amino acid sequence of PHS of S. antibioticus with other amino acid sequences indicated that PHS is a blue copper protein with copper binding domains in the N-terminal and C-terminal regions of the polypeptide chain. A BsrBI fragment containing the promoter region of phsA and a portion of the ORF was shown to promote xylE expression when cloned in the streptomycete promoter probe vector pIJ2843. This phsA promoter-dependent xylE expression could be repressed by glucose in S. antibioticus when the organism was grown on glucose or galactose plus glucose. Thus, the cloned promoter region appears to contain the sequences responsible for catabolite repression of PHS production.

“…These pioneering studies by Katz and Weissbach also demonstrated that the chromophore of actinomycin D, a phenoxazinone ring, is derived from the catabolism of tryptophan (19). Thus, 3-hydroxykynurenine is converted to 3-hydroxyanthranilic acid, and the latter intermediate is methylated to form 4-methyl-3-hydroxyanthranilic acid, the precursor of the phenoxazinone ring (28).…”

mentioning

confidence: 99%

Actinomycin Production Persists in a Strain of Streptomyces antibioticus Lacking Phenoxazinone Synthase

Antimicrob Agents Chemother

2000

Truncated fragments of the phenoxazinone synthase gene, phsA, were prepared by the PCR. The resulting fragments were cloned into conjugative plasmid pKC1132 and transferred to Streptomyces antibioticus by conjugation from Escherichia coli. Two of the resulting constructs were integrated into the S. antibioticus chromosome by homologous recombination, and each of the resulting strains, designated 3720/pJSE173 and 3720/pJSE174, contained a disrupted phsA gene. Strain 3720/pJSE173 grew poorly, and Southern blotting suggested that genetic changes other than the disruption of the phsA gene might have occurred during the construction of that strain. Strain 3720/pJSE174 sporulated well and grew normally on the medium used to prepare inocula for antibiotic production. Strain 3720/pJSE174 also grew as well as the wild-type strain on antibiotic production medium containing either 1 or 5.7 mM phosphate. Strain 3720/pJSE174 was shown to be devoid of phenoxazinone synthase (PHS) activity, and PHS protein was undetectable in this strain by Western blotting. Despite the absence of detectable PHS activity, strain 3720/pJSE174 produced slightly more actinomycin than did the wild-type parent strain in medium containing 1 or 5.7 mM phosphate. The observation that strain 3720/pJSE174, lacking detectable PHS protein or enzyme activity, retained the ability to produce actinomycin supports the conclusion that PHS is not required for actinomycin biosynthesis in S. antibioticus.The actinomycins are chromopeptide antibiotics produced by a number of Streptomyces strains and by some strains of Micromonospora (14,17). In actinomycin D, the pentapeptide chains contain several methylated amino acids and one Damino acid, but it was shown some years ago that the L-forms of the relevant amino acids serve as precursors for the synthesis of the forms found in the antibiotic (19). These pioneering studies by Katz and Weissbach also demonstrated that the chromophore of actinomycin D, a phenoxazinone ring, is derived from the catabolism of tryptophan (19). Thus, 3-hydroxykynurenine is converted to 3-hydroxyanthranilic acid, and the latter intermediate is methylated to form 4-methyl-3-hydroxyanthranilic acid, the precursor of the phenoxazinone ring (28).The enzymes required for the methylation of 3-hydroxyanthranilic acid, for the activation of the chromophore precursor, and for the activation of the amino acids in the pentapeptide chains and their incorporation into those chains have been isolated and characterized (7,22,23). Schauwecker and coworkers have recently cloned the gene cluster for actinomycin production from Streptomyces chrysomallus (27). Thus, many of the biochemical and molecular genetic details regarding the biosynthesis of actinomycin have been elucidated. One unanswered question regards the synthesis of the actinomycin chromophore. Some years ago, Katz and Weissbach (18) identified an enzyme in Streptomyces antibioticus that catalyzes the oxidative condensation of 2-aminophenol derivatives to produce phenoxazinones (Fig. 1). This en...