Identification of a monooxygenase from Streptomyces coelicolor A3(2) involved in biosynthesis of actinorhodin: purification and characterization of the recombinant enzyme

Abstract

The oxidation of phenols to quinones is an important reaction in the oxidative tailoring of many aromatic polyketides from bacterial and fungal systems. Sequence similarity between ActVA-Orf6 protein from the actinorhodin biosynthetic cluster and the previously characterized TcmH protein that is involved in tetracenomycin biosynthesis suggested that ActVA-Orf6 might catalyze this transformation as a step in actinorhodin biosynthesis. To investigate the role of ActVA-Orf6 in this oxidation, we have expressed the actVA-Orf6 gene in Escherichia coli and purified and characterized the recombinant protein. ActVA-Orf6 was shown to catalyze the monooxygenation of the tetracenomycin intermediate TcmF1 to TcmD3, strongly suggesting that it catalyzes oxidation of a similar intermediate in actinorhodin biosynthesis. The monooxygenase obeys simple reaction kinetics and has a K m of 4.8 ؎ 0.9 M, close to the figure reported for the homologous enzyme TcmH. The enzyme contains no prosthetic groups and requires only molecular oxygen to catalyze the oxidation. Site-directed mutagenesis was used to investigate the role of histidine residues thought to be important in the reaction; mutants lacking His-52 displayed much-reduced activity, consistent with the proposed mechanistic hypothesis that this histidine acts as a general base during catalysis.

Crystallization and preliminary X-ray diffraction studies of a monooxygenase fromStreptomyces coelicolorA3(2) involved in the biosynthesis of the polyketide actinorhodin

“…Our work has focused on the tailoring enzymes of these pathways, particularly those catalysing the oxidations that introduce hydroxy or quinone functionality to the polyketide molecules (Kendrew et al, 1995(Kendrew et al, , 1997. We have reported the expression, puri®cation and biochemical characterization of the enzyme ActVA-Orf6 (Kendrew et al, 1997), a monooxygenase from the actinorhodin pathway that catalyses the oxidation of phenolic compounds (or their keto tautomers) to the corresponding quinone (Fig.…”

“…We have reported the expression, puri®cation and biochemical characterization of the enzyme ActVA-Orf6 (Kendrew et al, 1997), a monooxygenase from the actinorhodin pathway that catalyses the oxidation of phenolic compounds (or their keto tautomers) to the corresponding quinone (Fig. 1).…”

“…ActVA-Orf6 monooxygenase was expressed and puri®ed from Escherichia coli following the methods previously described (Kendrew et al, 1997) with minor modi®cations. Puri®ed protein (approximately 98% pure as judged by Coomassie blue staining of an SDS±PAGE gel) was extensively dialysed against 10 mM Tris±HCl pH 8.0 and the concentration (8.2 mg ml À1 ) determined spectroscopically using a calculated extinction coef®cient (15 220 M À1 cm À1 at 280 nm).…”

“…The potential of this methodology is illustrated by numerous reports in which genetically engineered Streptomyces strains have been made to produce novel natural products whose structures have been rationally designed (McDaniel et al, 1995). This work has been aided by the cloning, sequence analysis and manipulation of the gene clusters responsible for the biosynthesis of aromatic and macrolide polyketides and has focused on the enzymes that catalyse the assembly and subsequent cyclization of the polyketide chain (for reviews, see Hopwood & Sherman, 1990;Hutchinson & Fujii, 1995;Hopwood, 1997;Hutchinson, 1997). This allowed many of the proteins responsible for the construction of the carbon skeleton to be identi®ed, but has left the identity and function of the enzymes that perform the later steps on these pathways relatively unclear.…”

Cloning of the biosynthetic gene cluster for naphthoxanthene antibiotic FD-594 from Streptomyces sp. TA-0256

“…22 Furthermore, PnxH showed less similarity to a cofactor-independent monooxygenase SnoaB (46%) in nogalamycin biosynthesis. 27 The crystal structure of SnoaB revealed that the completely conserved tryptophan residue in this family of antibiotic monooxygenases including ActVA-Orf6, 28,29 TcmH 30 and AknX 31 is catalytically important. 32 Indeed, the corresponding tryptophan residue is conserved in PnxH, supporting its involvement in quinone formation.…”