Thioesterases as tools for chemoenzymatic synthesis of macrolactones.

Macrocycles are a key functional group that can impart unique properties into molecules. Their synthesis has led to the development of many outstanding chemical methodologies and yet still remains challenging. Thioesterase (TE) domains are frequently responsible for macrocyclization in natural product biosynthesis and provide unique strengths for the enzymatic synthesis of macrocycles. In this feature article, we describe our work to characterize the substrate selectivity of TEs and to use these enzymes as biocatalysts. Our efforts have shown that the linear thioester activated substrates are loaded on TEs with limited substrate selectivity to generate acyl-enzyme intermediates. We show that cyclization of the acyl-enzyme intermediates can be highly selective, with competing hydrolysis of the acyl-enzyme intermediates. The mechanisms controlling TE-mediated macrocyclization versus hydrolysis are a significant unsolved problem in TE biochemistry. The potential of TEs as biocatalysts was demonstrated by using them in the chemoenzymatic total synthesis of macrocyclic depsipeptide natural products. This article highlights the strengths and potential of TEs as biocatalysts as well as their limitations, opening exciting research opportunities including TE engineering to optimize these powerful biocatalysts.

Total and chemoenzymatic synthesis of the lipodepsipeptide rhizomide A.

Rhizomides are a family of depsipeptide macrolactones synthesized by a non-ribosomal peptide synthetase (NRPS) encoded in the genome of Paraburkholderia rhizoxinica str. HKI 454. In this study, the total and chemoenzymatic synthesis of the depsipeptide rhizomide A is described. Rhizomide A was generated through macrolactamization while thelinear C-terminal N-acetylcysteamine (SNAC) thioester substrate was synthesized through a C-terminal thioesterification strategy. It was shown that the rhizomide A thioesterase (RzmA-TE) is an active macrocyclization catalyst, allowing the chemoenzymatic synthesis of rhizomide A.This work further showcases the biocatalytic power of TEs in accessing complex macrocyclic natural products.

In Vitro Biochemical Characterization of Excised Macrocyclizing Thioesterase Domains from Non-ribosomal Peptide Synthetases.

Characterization of thioesterases (TEs) is an important step in understanding natural product biosynthesis. Studying non-ribosomal peptide synthetase (NRPS) TEs presents a unique set of challenges with specific cloning and expression issues as well as the challenging synthesis of the thioester peptides substrate required for characterization of the TE. In this method, we describe the cloning and expression of NRPS TEs, the synthesis of thioester peptides, and the in vitro biochemical characterization of the enzyme.

Total and Chemoenzymatic Synthesis of Seongsanamide E.

The total and chemoenzymatic synthesis of the depsipeptide natural product seongsanamide E, 3, is described. The synthetic C-terminal N-acetylcysteamine thioester of linear natural product 1 was macrolactonized by the excised recombinant purified seongsanamide thioesterase (Sgd-TE) domain, generating 3. Sgd-TE also effects the ring opening of 3. Chemical synthesis provided 3 through a macrolactamization strategy. This work confirms the biosynthesis of 3 and demonstrates the power of Sgd-TE as a biocatalyst.

Armeniaspirol analogues with more potent Gram-positive antibiotic activity show enhanced inhibition of the ATP-dependent proteases ClpXP and ClpYQ.

Antibiotics with fundamentally new mechanisms of action such as the armeniaspirols, which target the ATP-dependent proteases ClpXP and ClpYQ, must be developed to combat antimicrobial resistance. While the mechanism of action of armeniaspirol against Gram-positive bacteria is understood, little is known about the structure-activity relationship for its antibiotic activity. Based on the preliminary data showing that modifications of armeniaspirol's N-methyl group increased antibiotic potency, we probed the structure-activity relationship of N-alkyl armeniaspirol derivatives. A series of focused derivatives were synthesized and evaluated for antibiotic activity against clinically relevant pathogens including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. Replacement of the N-methyl with N-hexyl, various N-benzyl, and N-phenethyl substituents led to substantial increases in antibiotic activity and potency for inhibition of both ClpYQ and ClpXP. Docking studies identified binding models for ClpXP and ClpYQ that were consistent with the inhibition data. This work confirms the role of ClpXP and ClpYQ in the mechanism of action of armeniaspirol and provides important lead compounds for further antibiotic development.

RpoN-Based stapled peptides with improved DNA binding suppress Pseudomonas aeruginosa virulence.

Stapled peptides have the ability to mimic α-helices involved in protein binding and have proved to be effective pharmacological agents for disrupting protein-protein interactions. DNA-binding proteins such as transcription factors bind their cognate DNA sequences via an α-helix interacting with the major groove of DNA. We previously developed a stapled peptide based on the bacterial alternative sigma factor RpoN capable of binding the RpoN DNA promoter sequence and inhibiting RpoN-mediated expression in Escherichia coli. We have elucidated a structure-activity relationship for DNA binding by this stapled peptide, improving DNA binding affinity constants in the high nM range. Lead peptides were shown to have low toxicity as determined by their low hemolytic activity at 100 µM and were shown to have anti-virulence activity in a Galleria mellonella model of Pseudomonas aeruginosa infection. These findings support further preclinical development of stapled peptides as antivirulence agents targeting P. aeruginosa.