Biosynthetic pathway toward carbohydrate-like moieties of alnumycins contains unusual steps for C-C bond formation and cleavage

Abstract

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 0 -C 2 0 bond of alnumycin C is subsequently cleaved and the ribose unit is rearranged into an unprecedented dioxolane (cisbicyclo[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.ribose-5-phosphate | natural product | biosynthesis | Streptomyces

Divergent evolution of an atypical S -adenosyl- l -methionine–dependent monooxygenase involved in anthracycline biosynthesis

“…The reactions with 8 lead to the accumulation of three products (A-C), whereas in the case of 9 only one product was observed (D). The columns present the formation of (A) 4-O-methyl-15-decarboxyaclacinomycin T (10), (B) 11-deoxy-β-rhodomycin T (11), (C) 4-O-methyl-11-deoxy-β-rhodomycin T (12), and (D) 11-deoxy-β-rhodomycin A (6) by the enzymes. The overall percentages may not add up to 100% in all samples because some unreacted substrate 15 was left with chimeras harboring poor activity (e.g., DnrK R2 + R3).…”

“…However, identification of the function of gene products residing in these pathways by bioinformatic analysis alone is complicated by the atypical chemistry the corresponding enzymes may catalyze. Unexpected chemical transformations appear to be especially common in the biosynthesis of aromatic polyketides, where biochemical studies have frequently revealed unusual reaction mechanisms (2,11), which includes instances where the reactivity of the substrates themselves are used in catalysis (12)(13)(14). In addition, homologous enzymes have been noted to catalyze diverse chemical transformations (15).…”

Effective Antibiofilm Polyketides against Staphylococcus aureus from the Pyranonaphthoquinone Biosynthetic Pathways of Streptomyces Species

“…Alnumycin A was isolated from the cultures of an S. albus strain that contained the intact alnumycin gene cluster on a cosmid pAlnuori. Other alnumycins were isolated from the deletion mutant strains S. albus/pAlnuori⌬ind (prealnumycin) (18), S. albus/ pAlnuori⌬aln3 (alnumycin B), and S. albus/pAlnuori⌬aln6 (alnumycins C1 and D) (19).…”

“…The alnumycins were isolated from the cultures of recombinant Streptomyces albus strains, purified to a minimum of 95% purity exactly as previously described (18,19), and stored in CHCl 3 at Ϫ20°C. Alnumycin A was isolated from the cultures of an S. albus strain that contained the intact alnumycin gene cluster on a cosmid pAlnuori.…”

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

“…Molecular genetic studies have clarified that the biosynthesis is initiated from a butyryl starter unit (46), while cyclization of the third ring is catalyzed by enzymes belonging to entirely different enzyme families than those found from the actinorhodin and granaticin pathways (35). Recently, the novel pathway leading to the attachment and formation of the sugar-like 4=-hydroxy-5=-hydroxymethyl-2=,7=-dioxane moiety was characterized in vivo and in vitro (34).…”