naphthoquinones and alnumycin

Covering: up to July 2018 Polyketides constitute a large family of natural products exhibiting various biological activities. Polyketide biosynthetic systems employ several strategies for the production of structurally diverse polyketides. Among the polyketide biosynthetic enzymes, a growing number of enzymes that catalyze a Michael-type addition have been identified. These enzymes are responsible for constructing unique polyketide backbone structures, forming heterocycles, and incorporating heteroatoms into the polyketide backbone, all of which contribute to the diversification of the polyketide structure. This review summarizes the current understanding of the function of enzymes catalyzing a Michael-type addition in polyketide biosynthesis, with a particular focus on mechanistic studies.

Topics: Bacteria; Biological Products; Catalysis; Ethers; Flavanones; Naphthoquinones; Polyketides; Rifamycins

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

Alnumycin A is an exceptional aromatic polyketide that contains a carbohydrate-like 4'-hydroxy-5'-hydroxymethyl-2',7'-dioxane moiety attached to the aglycone via a carbon-carbon bond. Recently, we have identified the D-ribose-5-phosphate origin of the dioxane unit and demonstrated that AlnA and AlnB are responsible for the overall C-ribosylation reaction. Here, we provide direct evidence that AlnA is a natural C-glycosynthase, which catalyzes the attachment of D-ribose-5-phosphate to prealnumycin by formation of the C(8)-C(1') bond as demonstrated by the structure of the intermediate alnumycin P. This compound is subsequently dephosphorylated by AlnB, an enzyme of the haloacid dehalogenase superfamily. Structure determination of the native trimeric AlnA to 2.1-Å resolution revealed a highly globular fold encompassing an α/β/α sandwich. The crystal structure of the complex with D-ribose-5-phosphate indicated that the phosphosugar is bound in the open-chain configuration. Identification of residues E29, K86, and K159 near the C-1 carbonyl of the ligand led us to propose that the carbon-carbon bond formation proceeds through a Michael-type addition. Determination of the crystal structure of the monomeric AlnB in the open conformation to 1.25-Å resolution showed that the protein consists of core and cap domains. Modeling of alnumycin P inside the cap domain positioned the phosphate group next to a Mg(2+) ion present at the junction of the domains. Mutagenesis data were consistent with the canonical reaction mechanism for this enzyme family revealing the importance of residues D15 and D17 for catalysis. The characterization of the prealnumycin C-ribosylation illustrates an alternative means for attachment of carbohydrates to natural products.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Biosynthetic Pathways; Catalysis; Crystallography, X-Ray; Glycosylation; Models, Biological; Models, Molecular; Molecular Sequence Data; Molecular Structure; Mutagenesis, Site-Directed; Naphthoquinones; Peptides; Polyketides; Ribosemonophosphates; Sequence Homology, Amino Acid; Static Electricity

Alnumycin A is an aromatic polyketide with a strong resemblance to related benzoisochromanequinone (BIQ) antibiotics, such as the model antibiotic actinorhodin. One intriguing difference between these metabolites is that the positions of the benzene and quinone rings are reversed in alnumycin A in comparison to the BIQ polyketides. In this paper we demonstrate that inactivation of either the monooxygenase alnT gene or the flavin reductase alnH gene results in the accumulation of a novel nonquinoid metabolite, thalnumycin A (ThA), in the culture medium. Additionally, two other previously characterized metabolites, K1115 A and 1,6-dihydroxy-8-propylanthraquinone (DHPA), were identified, which had oxidized into quinones putatively nonenzymatically at the incorrect position in the central ring. None of the compounds isolated contained correctly formed pyran rings, which suggests that on the alnumycin pathway quinone biosynthesis occurs prior to third ring cyclization. The regiochemistry of the two-component monooxygenase system AlnT/AlnH was finally confirmed in vitro by using ThA, FMN, and NADH in enzymatic synthesis, where the reaction product, thalnumycin B (ThB), was verified to contain the expected p-hydroquinone structure in the lateral ring.

Topics: Bacterial Proteins; Biosynthetic Pathways; Cyclization; Mixed Function Oxygenases; Molecular Structure; Naphthoquinones; Oxidation-Reduction; Quinones; Streptomyces

Carbohydrate moieties are important components of natural products, which are often imperative for the solubility and biological activity of the compounds. The aromatic polyketide alnumycin A contains an extraordinary sugar-like 4'-hydroxy-5'-hydroxymethyl-2',7'-dioxane moiety attached via a carbon-carbon bond to the aglycone. Here we have extensively investigated the biosynthesis of the dioxane unit through (13)C labeling studies, gene inactivation experiments and enzymatic synthesis. We show that AlnA and AlnB, members of the pseudouridine glycosidase and haloacid dehalogenase enzyme families, respectively, catalyze C-ribosylation conceivably through Michael-type addition of d-ribose-5-phosphate and dephosphorylation. The ribose moiety may be attached both in furanose (alnumycin C) and pyranose (alnumycin D) forms. The C(1')-C(2') bond of alnumycin C is subsequently cleaved and the ribose unit is rearranged into an unprecedented dioxolane (cis-bicyclo[3.3.0]-2',4',6'-trioxaoctan-3'β-ol) structure present in alnumycin B. The reaction is catalyzed by Aln6, which belongs to a previously uncharacterized enzyme family. The conversion was accompanied with consumption of O(2) and formation of H(2)O(2), which allowed us to propose that the reaction may proceed via hydroxylation of C1' followed by retro-aldol cleavage and acetal formation. Interestingly, no cofactors could be detected and the reaction was also conducted in the presence of metal chelating agents. The last step is the conversion of alnumycin B into the final end-product alnumycin A catalyzed by Aln4, an NADPH-dependent aldo-keto reductase. This characterization of the dioxane biosynthetic pathway sets the basis for the utilization of C-C bound ribose, dioxolane and dioxane moieties in the generation of improved biologically active compounds.

Topics: Bacterial Proteins; Biosynthetic Pathways; Carbohydrates; Carbon; Carbon Isotopes; Dioxanes; Electrophoresis, Polyacrylamide Gel; Glycoside Hydrolases; Hydrogen Peroxide; Hydrolases; Hydroxylation; Magnetic Resonance Spectroscopy; Molecular Structure; Naphthoquinones; Oxygen; Pseudouridine; Ribose; Ribosemonophosphates; Streptomyces

Benastatins are aromatic polyketides from Streptomyces spp. that efficiently inhibit glutathione-S-transferases and induce apoptosis. Their biosynthesis involves a type II polyketide synthase, and a ketoacyl synthase (KAS) III component (BenQ) similar to FabH that is crucial for providing and selecting the rare hexanoate PKS starter unit. The function of BenQ as a KAS III was experimentally proven by point mutation of the active site cysteine. In the mutant several novel short chain fatty acid derived penta- and hexacyclic benastatin derivatives with antiprolieferative activities are formed. Strategies for engineering benastatin biosynthesis were attempted. Synthetic starter units surrogates were not incorporated by block mutants, which suggests that the primer needs to be enzyme-bound. Thus, on the basis of KAS III crystal structures the three-dimensional structure of BenQ was modeled and the predicted substrate-binding tunnel was altered by individual mutations of potential gatekeeping residues (H95A and M99A). However, no significant changes in substrate specificity were observed, indicating that there are other or additional gatekeeping amino acid residues in BenQ or secondary factors including likely protein-protein interactions between BenQ and the PKS complex, and possible conformational changes in BenQ. Finally, a benQ null mutant was complemented with butyrate starter unit biosynthesis genes from the alnumycin biosynthesis gene cluster, which resulted in a great (10x) enhancement in the production of butyrate-primed hexacyclic benastatin derivatives. The successful generation of an alnumycin-benastatin FAS-PKS hybrid pathway highlights the potential of metabolic pathways, which may lead to novel potential therapeutics and increased yields of desired natural products.

Topics: Acyltransferases; Antineoplastic Agents; Cysteine; Escherichia coli; Glutathione Transferase; Mutation; Naphthacenes; Naphthoquinones; Point Mutation; Polyketide Synthases; Protein Binding; Protein Engineering; Protein Subunits; Substrate Specificity

Alnumycin is closely related to the benzoisochromanequinone (BIQ) polyketides such as actinorhodin. Exceptional structural features include differences in aglycone tailoring that result in the unique alnumycin chromophore and the existence of an unusual 4-hydroxymethyl-5-hydroxy-1,3-dioxan moiety. Cloning and sequencing of the alnumycin gene cluster from Streptomyces sp. CM020 revealed expected biosynthesis genes for polyketide assembly, but several genes encoding subsequent tailoring enzymes were highly atypical. Heterologous expression studies confirmed that all of the genes required for alnumycin biosynthesis resided within the sequenced clone. Inactivation of genes aln4 and aln5 showed that the mechanism of pyran ring formation differs from actinorhodin and granaticin pathways. Further inactivation studies identified two genes, alnA and alnB, involved in the synthesis and attachment of the dioxan moiety, and resulted in the production of the polyketide prealnumycin.

Topics: Cloning, Molecular; Dioxanes; Gene Expression; Genome, Fungal; Macrolides; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Molecular Structure; Multigene Family; Naphthoquinones; Pyrans; Streptomyces

Topics: Anti-Bacterial Agents; Naphthoquinones; Streptomyces