Epoxyquinone Formation Catalyzed by a Two‐Component Flavin‐Dependent Monooxygenase Involved in Biosynthesis of the Antibiotic Actinorhodin

Abstract

The biosynthetic gene cluster of the aromatic polyketide antibiotic actinorhodin (ACT) in Streptomyces coelicolor A3(2) carries a pair of genes, actVA-ORF5 and actVB, that encode a two-component flavin-dependent monooxygenase (FMO). Our previous studies have demonstrated that the ActVA-ORF5/ActVB system functions as a quinone-forming C-6 oxygenase in ACT biosynthesis. Furthermore, we found that this enzyme system exhibits an additional oxygenation activity with dihydrokalafungin (DHK), a proposed intermediate in the ACT biosynthetic pathway, and generates two reaction products. These compounds were revealed to be monooxygenated derivatives of kalafungin, which is spontaneously formed through oxidative lactonization of DHK. Their absolute structures were elucidated from their NMR spectroscopic data and by computer modeling and X-ray crystallography as (5S,14R)-epoxykalafungin and (5R,14S)-epoxykalafungin, demonstrating an additional epoxyquinone-forming activity of the ActVA-ORF5/ActVB system in vitro.

Protein expression analysis of a high-demeclocycline producing strain of Streptomyces aureofaciens and the roles of CtcH and CtcJ in demeclocycline biosynthesis

“…These oxygenases belong to the so-called post-PKS-modifying (tailoring) enzymes, which play crucial roles in the formation of interesting and unique molecular structures. The monooxygenases are representative, tailoring enzymes that are especially important in providing the structural elements essential for the special biological activity of these structures (Taguchi et al 2011;Faust et al 2000).…”

“…Above all, oxidation reactions catalyzed by monooxygenases are necessary for the formation of 6-DCT ( Fig. 1) (Taguchi et al 2011;Bentley et al 2002).…”

Bifunctionality of ActIV as a Cyclase‐Thioesterase Revealed by in Vitro Reconstitution of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2)

“…(S)-DNPAi s produced by stereospecific pyranr ing formation:C -3 reduction, hemiketal formation, andd ehydration at C-6/15. [20] Enzymatic formationo f( S)-DNPAf rom Xd emonstrates the bifunctionality of ActIV as aC YC-TE Based on the biosynthetic scheme ( Figure 1A), 8b is asubstrate of RED1, which produces 9.I nv itro reaction mixtures of the ActIV system ( Figure 5A)w ere subjected to gel-filtration to collect the fraction containing X,f ollowed by HPLC analysis (Figure 5B). Consistent with our previous study,t he presence of Y might reflect the converting property of dihydro-forms of BIQ to lactone during biochemical characterization.…”

Characterization of the Two-Component Monooxygenase System AlnT/AlnH Reveals Early Timing of Quinone Formation in Alnumycin Biosynthesis

“…The sequence identified with highest similarity was a putative oxygenase (GenBank accession number AEM44243.1) from an uncultured bacterium (54% identity). More relevant in terms of putative function were the angucyclinone hydroxylase LndZ5 (37) from Streptomyces globisporus (53% identity) and ActVA-ORF5 (36,(42)(43)(44) from Streptomyces coelicolor A3(2) (48% identity), involved in biosynthesis of the aromatic polyketides landomycin and actinorhodin, respectively. Sequence comparison with the monooxygenase component of p-hydroxyphenylacetate hydroxylase (see Fig.…”

“…The alnumycin biosynthetic cluster (35), on the other hand, only contains genes for the putative two-component mono-oxygenase system, alnT and alnH, which encode a flavin-dependent monooxygenase and a flavin reductase, respectively. The AlnT/AlnH pair is homologous to the ActVA-ORF5/ActVB from the actinorhodin pathway, respectively, which has been studied in recent years using molecular genetics (36) and biochemistry (42)(43)(44). The lack of a cofactorless oxygenase homologous to ActVA-ORF6 on the alnumycin pathway suggests a closer evolutionary relationship to the granaticin (20) and medermycin (21) (Fig.…”