Brian J. Beck

Generation of multiple bioactive macrolides by hybrid modular polyketide synthases in Streptomyces venezuelae.

The plasmid-based replacement of the multifunctional protein subunits of the pikromycin PKS in S. venezuelae by the corresponding subunits from heterologous modular PKSs resulted in recombinant strains that produce both 12- and 14-membered ring macrolactones with predicted structural alterations. In all cases, novel macrolactones were produced and further modified by the DesVII glycosyltransferase and PikC hydroxylase, leading to biologically active macrolide structures. These results demonstrate that hybrid PKSs in S. venezuelae can produce a multiplicity of new macrolactones that are modified further by the highly flexible DesVII glycosyltransferase and PikC hydroxylase tailoring enzymes. This work demonstrates the unique capacity of the S. venezuelae pikromycin pathway to expand the toolbox of combinatorial biosynthesis and to accelerate the creation of novel biologically active natural products.

Biochemical Evidence for an Editing Role of Thioesterase II in the Biosynthesis of the Polyketide Pikromycin

The pikromycin biosynthetic gene cluster contains the pikAV gene encoding a type II thioesterase (TEII). TEII is not responsible for polyketide termination and cyclization, and its biosynthetic role has been unclear. During polyketide biosynthesis, extender units such as methylmalonyl acyl carrier protein (ACP) may prematurely decarboxylate to generate the corresponding acyl-ACP, which cannot be used as a substrate in the condensing reaction by the corresponding ketosynthase domain, rendering the polyketide synthase module inactive. It has been proposed that TEII may serve as an “editing” enzyme and reactivate these modules by removing acyl moieties attached to ACP domains. Using a purified recombinant TEII we have tested this hypothesis by using in vitro enzyme assays and a range of acyl-ACP, malonyl-ACP, and methylmalonyl-ACP substrates derived from either PikAIII or the loading didomain of DEBS1 (6-deoxyerythronolide B synthase; ATL-ACPL). The pikromycin TEII exhibited highK m values (>100 μm) with all substrates and no apparent ACP specificity, catalyzing cleavage of methylmalonyl-ACP from both ATL-ACPL(k cat/K m 3.3 ± 1.1m −1 s−1) and PikAIII (k cat/K m 2.9 ± 0.9m −1 s−1). The TEII exhibited some acyl-group specificity, catalyzing hydrolysis of propionyl (k cat/K m 15.8 ± 1.8m −1 s−1) and butyryl (k cat/K m 17.5 ± 2.1m −1 s−1) derivatives of ATL-ACPL faster than acetyl (k cat/K m 4.9 ± 0.7m −1 s−1), malonyl (k cat/K m 3.9 ± 0.5m −1 s−1), or methylmalonyl derivatives. PikAIV containing a TEI domain catalyzed cleavage of propionyl derivative of ATL-ACPL at a dramatically lower rate than TEII. These results provide the first unequivocal in vitro evidence that TEII can hydrolyze acyl-ACP thioesters and a model for the action of TEII in which the enzyme remains primarily dissociated from the polyketide synthase, preferentially removing aberrant acyl-ACP species with long half-lives. The lack of rigorous substrate specificity for TEII may explain the surprising observation that high level expression of the protein inStreptomyces venezuelae leads to significant (>50%) titer decreases.

The 1.92 A Structure of Streptomyces Coelicolor A3(2) Cyp154C1: A New Monooxygenase that Functionalizes Macrolide Ring Systems

Evolutionary links between cytochrome P450 monooxygenases, a superfamily of extraordinarily divergent heme-thiolate proteins catalyzing a wide array of NADPH/NADH- and O2-dependent reactions, are becoming better understood because of availability of an increasing number of fully sequenced genomes. Among other reactions, P450s catalyze the site-specific oxidation of the precursors to macrolide antibiotics in the genus Streptomyces introducing regiochemical diversity into the macrolide ring system, thereby significantly increasing antibiotic activity. Developing effective uses forStreptomyces enzymes in biosynthetic processes and bioremediation requires identification and engineering of additional monooxygenases with activities toward a diverse array of small molecules. To elucidate the molecular basis for substrate specificity of oxidative enzymes toward macrolide antibiotics, the x-ray structure of CYP154C1 from Streptomyces coelicolor A3(2) was determined (Protein Data Bank code 1GWI). Relocation of certain common P450 secondary structure elements, along with a novel structural feature involving an additional β-strand transforming the five-stranded β-sheet into a six-stranded variant, creates an open cleft-shaped substrate-binding site between the two P450 domains. High sequence similarity to macrolide monooxygenases from other microbial species translates into catalytic activity of CYP154C1 toward both 12- and 14-membered ring macrolactonesin vitro.

The hidden steps of domain skipping: macrolactone ring size determination in the pikromycin modular polyketide synthase.

The pikromycin (Pik) polyketide synthase (PKS) from Streptomyces venezuelae comprises four multifunctional polypeptides (PikAI, PikAII, PikAIII, and PikAIV). This PKS can generate 12- and 14-membered ring macrolactones (10-deoxymethynolide and narbonolide, respectively) through the activity of its terminal modules (PikAIII and PikAIV). We performed a series of experiments involving the functional replacement of PikAIV in mutant strains with homodimeric and heterodimeric PikAIV modules to investigate the details of macrolactone ring size determination. The results suggest a new and surprising mechanism by which the penultimate hexaketide chain elongation intermediate is transferred from PikAIII ACP5 to PikAIV ACP6 before release by the terminal thioesterase domain. Elucidation of this chain transfer mechanism provides important new details about alternative macrolactone ring size formation in modular PKSs and contributes to the potential for rational design of structural diversity by combinatorial biosynthesis.

Aryl Acid Adenylating Enzymes Involved in Siderophore Biosynthesis: Fluorescence Polarization Assay, Ligand Specificity, and Discovery of Non-nucleoside Inhibitors via High-Throughput Screening

The design and synthesis of a fluorescent probe Fl-Sal-AMS 6 based on the tight-binding inhibitor 5- O-[ N-(salicyl)sulfamoyl]adenosine (Sal-AMS) is described for the aryl acid adenylating enzymes (AAAEs) known as MbtA, YbtE, EntE, VibE, DhbE, and BasE involved in siderophore biosynthesis from Mycobacterium tuberculosis, Yersinia pestis, Escherichia coli, Vibrio cholerae, Bacillus subtilis, and Acinetobacter baumannii, respectively. The probe was successfully used to develop a fluorescence polarization assay for these six AAAEs, and equilibrium dissociation constants were determined in direct binding experiments. Fl-Sal-AMS was effective for AAAEs that utilize salicylic acid or 2,3-dihydroxybenzoic acid as native substrates, with dissociation constants ranging from 9-369 nM, but was ineffective for AsbC, the AAAE from Bacillus anthracis, which activates 3,4-dihydroxybenzoic acid. Competitive binding experiments using a series of ligands including substrates, reaction products, and inhibitors provided the first comparative structure-activity relationships for AAAEs. The fluorescence polarization assay was then miniaturized to a 384-well plate format, and high-throughput screening was performed at the National Screening Laboratory for the Regional Centers of Excellence in Biodefense and Emerging Infectious Diseases (NSRB) against BasE, an AAAE from Acinetobacter baumannii involved in production of the siderophore acinetobactin. Several small molecule inhibitors with new chemotypes were identified, and compound 23 containing a pyrazolo[5,4- a]pyridine scaffold emerged as the most promising ligand with a K D of 78 nM, which was independently confirmed by isothermal calorimetry, and inhibition was also verified in an ATP-[ (32)P]-pyrophosphate exchange steady-state kinetic assay.

Recent developments in the production of novel polyketides by combinatorial biosynthesis.

Polyketides are a class of structurally diverse natural products which possess a wide range of biological activities (Hopwood, 1997). These compounds are used throughout medicine and agriculture as antimicrobials, immunosuppressants, antiparasitics, and anticancer agents. While structurally diverse, poIyketides are assembled by a common mechanism of decarboxylative condensations of simple malonate derivatives by polyketide synthases (PKSs) in a manner very similar to fatty acid biosynthesis. After assembly by the PKS, tailoring enzymes such as glycosyltransferases, hydroxylases, or methyltransferases can then further modify the polyketide product. These post-PKS modifications are almost always necessary in order for the molecule to be bioactive. It has been shown that, frequently, bacteria (primarily the actinomycetes) possess both multifunctional (type!) and multicomponent (type H) PKSs. The first type I PKS to be sequenced and characterized was 6-deoxyerythronolide B synthase (DEBS) from Saccharopolyspora etythraea, which produces the polyketide macrolactone ring of erythromycin (Figure 8.1) (Cortes et al., 1990; Donadio a al., 1991; Caffrey et al., 1992). This modular PKS contains a loading module responsible for selecting a primer unit (in this case, propionyl CoA) and six extension modules. It was shown