alnumycin and actinorhodin

Proteins belonging to the NTF2-like superfamily are present in the biosynthetic pathways of numerous polyketide natural products, such as anthracyclins and benzoisochromanequinones. Some have been found to be bona fide polyketide cyclases, but many of them have roles that are currently unknown. Here, the X-ray crystal structures of three NTF2-like proteins of unknown function are reported: those of ActVI-ORFA from Streptomyces coelicolor A3(2) and its homologs Caci_6494, a protein from an uncharacterized biosynthetic cluster in Catenulispora acidiphila, and Aln2 from Streptomyces sp. CM020, a protein in the biosynthetic pathway of alnumycin. The presence of a solvent-accessible cavity and the conservation of the His/Asp dyad that is characteristic of many polyketide cyclases suggest a potential enzymatic role for these enzymes in polyketide biosynthesis.

Topics: Actinobacteria; Amino Acid Sequence; Anthraquinones; Bacterial Proteins; Binding Sites; Cloning, Molecular; Crystallography, X-Ray; Escherichia coli; Gene Expression; Genetic Vectors; Models, Molecular; Multigene Family; Naphthoquinones; Polyketides; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Recombinant Proteins; Streptomyces; Streptomyces coelicolor; Substrate Specificity

Actinorhodin (ACT) produced by Streptomyces coelicolor A3(2) belongs to the benzoisochromanequinone (BIQ) class of antibiotics. ActVA-ORF5, a flavin-dependent monooxygenase (FMO) essential for ACT biosynthesis, forms a two-component enzyme system in combination with a flavin:NADH oxidoreductase, ActVB. The genes for homologous two-component FMOs are found in the biosynthetic gene clusters for two other BIQs, granaticin (GRA) and medermycin (MED), and a closely related antibiotic, alnumycin (ALN). Our functional analysis of these FMOs (ActVA-ORF5, Gra-ORF21, Med-ORF7, and AlnT) in S. coelicolor unambiguously demonstrated that ActVA-ORF5 and Gra-ORF21 are bifunctional and capable of both p-quinone formation at C-6 in the central ring and C-8 hydroxylation in the lateral ring, whereas Med-ORF7 catalyzes only p-quinone formation. No p-quinone formation on a BIQ substrate was observed for AlnT, which is involved in lateral p-quinone formation in ALN.

Topics: Anthraquinones; Anti-Bacterial Agents; Bacterial Proteins; Chromatography, High Pressure Liquid; Mixed Function Oxygenases; Multigene Family; NADH, NADPH Oxidoreductases; Naphthoquinones; Quinones; Recombinant Proteins; Spectrometry, Mass, Electrospray Ionization; Streptomyces