Molecular basis for control of antibiotic production by a bacterial hormone.

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.

Structure and function of the RedJ protein, a thioesterase from the prodiginine biosynthetic pathway in Streptomyces coelicolor.

Prodiginines are a class of red-pigmented natural products with immunosuppressant, anticancer, and antimalarial activities. Recent studies on prodiginine biosynthesis in Streptomyces coelicolor have elucidated the function of many enzymes within the pathway. However, the function of RedJ, which was predicted to be an editing thioesterase based on sequence similarity, is unknown. We report here the genetic, biochemical, and structural characterization of the redJ gene product. Deletion of redJ in S. coelicolor leads to a 75% decrease in prodiginine production, demonstrating its importance for prodiginine biosynthesis. RedJ exhibits thioesterase activity with selectivity for substrates having long acyl chains and lacking a ß-carboxyl substituent. The thioesterase has 1000-fold greater catalytic efficiency with substrates linked to an acyl carrier protein (ACP) than with the corresponding CoA thioester substrates. Also, RedJ strongly discriminates against the streptomycete ACP of fatty acid biosynthesis in preference to RedQ, an ACP of the prodiginine pathway. The 2.12 Å resolution crystal structure of RedJ provides insights into the molecular basis for the observed substrate selectivity. A hydrophobic pocket in the active site chamber is positioned to bind long acyl chains, as suggested by a long-chain ligand from the crystallization solution bound in this pocket. The accessibility of the active site is controlled by the position of a highly flexible entrance flap. These data combined with previous studies of prodiginine biosynthesis in S. coelicolor support a novel role for RedJ in facilitating transfer of a dodecanoyl chain from one acyl carrier protein to another en route to the key biosynthetic intermediate 2-undecylpyrrole.

Stereochemical elucidation of streptorubin B.

Streptorubin B is a structurally remarkable member of the prodiginine group of antibiotics produced by several actinobacteria, including the model organism Streptomyces coelicolor A3(2). Transannular strain within the pyrrolophane structure of this molecule causes restricted rotation that gives rise to the possibility of (diastereomeric) atropisomers. Neither the relative nor the absolute stereochemistry of streptorubin B is known. NOESY NMR experiments were used to define the relative stereochemistry of the major atropisomer of streptorubin B·HCl in solution as anti. We exploited this finding together with our knowledge of streptorubin B biosynthesis in S. coelicolor to determine the absolute stereochemistry of the anti atropisomer. 2-Undecylpyrrole stereoselectively labeled with deuterium at C-4' was synthesized and fed to a mutant of S. coelicolor, which was unable to produce streptorubin B because it was blocked in 2-undecylpyrrole biosynthesis, and in which the genes responsible for the last two steps of streptorubin B biosynthesis were overexpressed. (1)H and (2)H NMR analysis of the stereoselectively deuterium-labeled streptorubin B·HCl produced by this mutasynthesis strategy allowed us to assign the absolute stereochemistry of the major (anti) atropisomer as 7'S. HPLC analyses of streptorubin B isolated from S. coelicolor on a homochiral stationary phase and comparisons with streptorubin B derived from an enantioselective synthesis showed that the natural product consists of an approximately 88:7:5 mixture of the (7'S, anti), (7'S, syn), and (7'R, anti) stereoisomers.

Elucidation of the Streptomyces coelicolor pathway to 2-undecylpyrrole, a key intermediate in undecylprodiginine and streptorubin B biosynthesis.

The red gene cluster of Streptomyces coelicolor directs production of undecylprodiginine. Here we report that this gene cluster also directs production of streptorubin B and show that 2-undecylpyrrole (UP) is an intermediate in the biosynthesis of undecylprodiginine and streptorubin B. The redPQRKL genes are involved in UP biosynthesis. RedL and RedK are proposed to generate UP from dodecanoic acid or a derivative. A redK(-) mutant produces a hydroxylated undecylprodiginine derivative, whereas redL(-) and redK(-) mutants require addition of chemically synthesized UP for production of undecylprodiginine and streptorubin B. Fatty acid biosynthetic enzymes can provide dodecanoic acid, but efficient and selective prodiginine biosynthesis requires RedPQR. Deletion of redP, redQ, or redR leads to an 80%-95% decrease in production of undecylprodiginine and an array of prodiginine analogs with varying alkyl chains. In a redR(-) mutant, the ratio of these can be altered in a logical manner by feeding various fatty acids.

A dual transacylation mechanism for polyketide synthase chain release in enacyloxin antibiotic biosynthesis.

Polyketide synthases assemble diverse natural products with numerous important applications. The thioester intermediates in polyketide assembly are covalently tethered to acyl carrier protein domains of the synthase. Several mechanisms for polyketide chain release are known, contributing to natural product structural diversification. Here, we report a dual transacylation mechanism for chain release from the enacyloxin polyketide synthase, which assembles an antibiotic with promising activity against Acinetobacter baumannii. A non-elongating ketosynthase domain transfers the polyketide chain from the final acyl carrier protein domain of the synthase to a separate carrier protein, and a non-ribosomal peptide synthetase condensation domain condenses it with (1S,3R,4S)-3,4-dihydroxycyclohexane carboxylic acid. Molecular dissection of this process reveals that non-elongating ketosynthase domain-mediated transacylation circumvents the inability of the condensation domain to recognize the acyl carrier protein domain. Several 3,4-dihydroxycyclohexane carboxylic acid analogues can be employed for chain release, suggesting a promising strategy for producing enacyloxin analogues.

Structural basis for chain release from the enacyloxin polyketide synthase.

Modular polyketide synthases and non-ribosomal peptide synthetases are molecular assembly lines that consist of several multienzyme subunits that undergo dynamic self-assembly to form a functional megacomplex. N- and C-terminal docking domains are usually responsible for mediating the interactions between subunits. Here we show that communication between two non-ribosomal peptide synthetase subunits responsible for chain release from the enacyloxin polyketide synthase, which assembles an antibiotic with promising activity against Acinetobacter baumannii, is mediated by an intrinsically disordered short linear motif and a ß-hairpin docking domain. The structures, interactions and dynamics of these subunits were characterized using several complementary biophysical techniques to provide extensive insights into binding and catalysis. Bioinformatics analyses reveal that short linear motif/ß-hairpin docking domain pairs mediate subunit interactions in numerous non-ribosomal peptide and hybrid polyketide-non-ribosomal peptide synthetases, including those responsible for assembling several important drugs. Short linear motifs and ß-hairpin docking domains from heterologous systems are shown to interact productively, highlighting the potential of such interfaces as tools for biosynthetic engineering.

Role and substrate specificity of the Streptomyces coelicolor RedH enzyme in undecylprodiginine biosynthesis.

The function of RedH from Streptomyces coelicolor as an enzyme that catalyses the condensation of 4-methoxy-2,2'-bipyrrole-5-carboxaldehyde (MBC) and 2-undecylpyrrole to form the natural product undecylprodiginine has been experimentally proven, and the substrate specificity of RedH has been probed in vivo by examining its ability to condense chemically-synthesised MBC analogues with 2-undecylpyrrole to afford undecylprodiginine analogues.

Oxidative tailoring reactions catalyzed by nonheme iron-dependent enzymes: streptorubin B biosynthesis as an example.

Tailoring enzymes catalyze reactions that modify natural product backbone structures before, during, or after their biosynthesis to create a final product with specific biological activities. Such reactions can be catalyzed by a myriad of different enzyme families and are responsible for a wide variety of transformations including regio- and/or stereospecific acylation, alkylation, glycosylation, halogenation, and oxidation. Within a broad group of oxidative tailoring enzymes, there is a rapidly growing family of nonheme iron- and oxygen-dependent enzymes that catalyze a variety of remarkable hydroxylation, desaturation, halogenation, and oxidative cyclization reaction in the biosynthesis of several important metabolites, including carbapenems, penicillins, cephalosporins, clavams, prodiginines, fosfomycin, syringomycin, and coronatine. In this chapter, we report an expedient method for analyzing tailoring enzymes that catalyze oxidative cyclization reactions in prodiginine biosynthesis via expression of the corresponding genes in a heterologous host, feeding of putative biosynthetic intermediates to the resulting strains, and liquid chromatography-mass spectrometry analyses of the metabolites produced.

Regio- and stereodivergent antibiotic oxidative carbocyclizations catalysed by Rieske oxygenase-like enzymes.

Oxidative cyclizations, exemplified by the biosynthetic assembly of the penicillin nucleus from a tripeptide precursor, are arguably the most synthetically powerful implementation of C-H activation reactions in nature. Here, we show that Rieske oxygenase-like enzymes mediate regio- and stereodivergent oxidative cyclizations to form 10- and 12-membered carbocyclic rings in the key steps of the biosynthesis of the antibiotics streptorubin B and metacycloprodigiosin, respectively. These reactions represent the first examples of oxidative carbocyclizations catalysed by non-haem iron-dependent oxidases and define a novel type of catalytic activity for Rieske enzymes. A better understanding of how these enzymes achieve such remarkable regio- and stereocontrol in the functionalization of unactivated hydrocarbon chains will greatly facilitate the development of selective man-made C-H activation catalysts.