ABSTRACT
Actinobacteria produce numerous antibiotics and other specialized metabolites that have important applications in medicine and agriculture1. Diffusible hormones frequently control the production of such metabolites by binding TetR family transcriptional repressors (TFTRs), but the molecular basis for this remains unclear2. The production of methylenomycin antibiotics in Streptomyces coelicolor A3(2) is initiated by the binding of 2-alkyl-4-hydroxymethylfuran-3-carboxylic acid (AHFCA) hormones to the TFTR MmfR3. Here we report the X-ray crystal structure of an MmfR-AHFCA complex, establishing the structural basis for hormone recognition. We also elucidate the mechanism for DNA release upon hormone binding through the single-particle cryo-electron microscopy structure of an MmfR-operator complex. DNA binding and release assays with MmfR mutants and synthetic AHFCA analogues define the role of individual amino acid residues and hormone functional groups in ligand recognition and DNA release. These findings will facilitate the exploitation of actinobacterial hormones and their associated TFTRs in synthetic biology and in the discovery of new antibiotics.
Subject(s)
Anti-Bacterial Agents/biosynthesis , Furans/metabolism , Streptomyces coelicolor/metabolism , Apoproteins/chemistry , Apoproteins/metabolism , Apoproteins/ultrastructure , Bacterial Proteins/chemistry , Bacterial Proteins/classification , Bacterial Proteins/metabolism , Bacterial Proteins/ultrastructure , Cryoelectron Microscopy , Crystallography, X-Ray , DNA/chemistry , DNA/genetics , DNA/metabolism , DNA/ultrastructure , Furans/chemistry , Hormones/chemistry , Hormones/classification , Hormones/metabolism , Ligands , Models, Molecular , Peptides/metabolism , Repressor Proteins/chemistry , Repressor Proteins/classification , Repressor Proteins/metabolism , Repressor Proteins/ultrastructure , Signal Transduction , Streptomyces coelicolor/chemistry , Streptomyces coelicolor/genetics , Structure-Activity RelationshipABSTRACT
Using a combination of a synthetic substrate analogue and product standard, MmfL, a homologue of the γ-butyrolactone biosynthetic enzyme AfsA, was shown to catalyse the condensation of dihydroxyacetone phosphate with a ß-ketoacyl thioester to form a phosphorylated butenolide intermediate in the biosynthesis of the methylenomycin furans, which induce methlenomycin antibiotic production in Streptomyces coelicolor A3(2). AfsA homologues are also involved in the biosynthesis of 2-akyl-4-hydroxy-3-methyl butenolide inducers of antibiotic production in other Streptomyces species, indicating that diverse signalling molecules are assembled from analogous phosphorylated butenolide intermediates.
Subject(s)
4-Butyrolactone/analogs & derivatives , Anti-Bacterial Agents/chemistry , Bacterial Proteins/chemistry , Furans/chemistry , 4-Butyrolactone/chemistry , Biosynthetic Pathways , Catalysis , Escherichia coli , Gene Expression Regulation, Bacterial , Peptides/chemistry , Peptides/metabolism , Phosphorylation , Streptomyces , Structure-Activity RelationshipABSTRACT
The label from [3-(13)C]-L-glycerol is incorporated into the hydroxymethyl group of methylenomycin furans suggesting a butenolide intermediate in their biosynthesis.
