Adenylation Domain-Guided Recruitment of Trans-Acting Nonheme Monooxygenases in Nonribosomal Peptide Biosynthesis.

Nonheme diiron monooxygenases (NHDMs) interact with nonribosomal peptide synthetase (NRPS) assembly lines to install ß-hydroxylations at thiolation-domain-bound amino acids during nonribosomal peptide biosynthesis. The high potential of this enzyme family to diversify the products of engineered assembly lines is disproportionate to the currently small knowledge about their structures and mechanisms of substrate recognition. Here, we report the crystal structure of FrsH, the NHDM which catalyzes the ß-hydroxylation of l-leucines during biosynthesis of the depsipeptide G protein inhibitor FR900359. Using biophysical approaches, we provide evidence that FrsH interacts with the cognate monomodular NRPS FrsA. By AlphaFold modeling and mutational studies, we detect and examine structural features within the assembly line crucial to recruit FrsH for leucine ß-hydroxylation. These are, in contrast to cytochrome-dependent NRPS ß-hydroxylases, not located on the thiolation domain, but on the adenylation domain. FrsH can be functionally substituted by homologous enzymes from biosyntheses of the cell-wall-targeting antibiotics lysobactin and hypeptin, indicating that these features are generally applicable to members of the family of trans-acting NHDMs. These insights give important directions for the construction of artificial assembly lines to yield bioactive and chemically complex peptide products.

Carrier Protein-Free Enzymatic Biaryl Coupling in Arylomycin A2 Assembly and Structure of the Cytochrome P450 AryC.

Invited for the cover of this issue are Sabine Schneider, Tobias A. M. Gulder and co-workers at Technical University of Dresden, Technical University of Munich and Ludwig-Maximillians-University Munich. The image depicts the crystal structure of the cytochrome P450 AryC from arylomycin biosynthesis. Read the full text of the article at 10.1002/chem.202103389.

A Specialized Dehydrogenase Provides l-Phenyllactate for FR900359 Biosynthesis.

d-Phenyllactate (PLA) is a component of the selective Gq protein inhibitor and nonribosomal cyclic depsipeptide FR900359 (FR). Here we report a detailed biochemical investigation of PLA biosynthesis and its incorporation into the natural product FR. The enzyme FrsC, member of the lactate/malate dehydrogenase superfamily, was shown to catalyze the formation of l-PLA from phenylpyruvate. FrsC was kinetically characterized and its substrate specificity determined. Incorporation of l-PLA was probed by assaying the adenylation domain FrsE-A3 and feeding studies with a Chromobacterium vaccinii ΔfrsC mutant, confirming preferred activation of l-PLA followed by on-line epimerization to d-PLA. Finally, detailed bioinformatic analyses of FrsC revealed its close relation to malate dehydrogenases from primary metabolism and suggest extensions in the substrate binding loop to be responsible for its adaptation to accepting larger aromatic substrates with high specificity.

Carrier Protein-Free Enzymatic Biaryl Coupling in Arylomycin A2 Assembly and Structure of the Cytochrome P450 AryC.

The arylomycin antibiotics are potent inhibitors of bacterial type I signal peptidase. These lipohexapeptides contain a biaryl structural motif reminiscent of glycopeptide antibiotics. We herein describe the functional and structural evaluation of AryC, the cytochrome P450 performing biaryl coupling in biosynthetic arylomycin assembly. Unlike its enzymatic counterparts in glycopeptide biosynthesis, AryC converts free substrates without the requirement of any protein interaction partner, likely enabled by a strongly hydrophobic cavity at the surface of AryC pointing to the substrate tunnel. This activity enables chemo-enzymatic assembly of arylomycin A2 that combines the advantages of liquid- and solid-phase peptide synthesis with late-stage enzymatic cross-coupling. The reactivity of AryC is unprecedented in cytochrome P450-mediated biaryl construction in non-ribosomal peptides, in which peptidyl carrier protein (PCP)-tethering so far was shown crucial both in vivo and in vitro.

Thioesterase-mediated side chain transesterification generates potent Gq signaling inhibitor FR900359.

The potent and selective Gq protein inhibitor depsipeptide FR900359 (FR), originally discovered as the product of an uncultivable plant endosymbiont, is synthesized by a complex biosynthetic system comprising two nonribosomal peptide synthetase (NRPS) assembly lines. Here we characterize a cultivable bacterial FR producer, enabling detailed investigations into biosynthesis and attachment of the functionally important FR side chain. We reconstitute side chain assembly by the monomodular NRPS FrsA and the non-heme monooxygenase FrsH, and characterize intermolecular side chain transesterification to the final macrocyclic intermediate FR-Core, mediated by the FrsA thioesterase domain. We harness FrsA substrate promiscuity to generate FR analogs with altered side chains and demonstrate indispensability of the FR side chain for efficient Gq inhibition by comparative bioactivity, toxicity and docking studies. Finally, evolution of FR and side chain biosynthesis is discussed based on bioinformatics analyses. Side chain transesterification boosts potency and target affinity of selective Gq inhibitor natural products.

Heterologous Expression, Biosynthetic Studies, and Ecological Function of the Selective Gq-Signaling Inhibitor FR900359.

The cyclic depsipeptide FR900359 (FR), isolated from the tropical plant Ardisia crenata, is a strong and selective inhibitor of Gq proteins, making it an indispensable pharmacological tool to study Gq-related processes, as well as a promising drug candidate. Gq inhibition is a novel mode of action for defense chemicals and crucial for the ecological function of FR, as shown by in vivo experiments in mice, its affinity to insect Gq proteins, and insect toxicity studies. The uncultured endosymbiont of A. crenata was sequenced, revealing the FR nonribosomal peptide synthetase (frs) gene cluster. We here provide a detailed model of FR biosynthesis, supported by in vitro enzymatic and bioinformatic studies, and the novel analogue AC-1, which demonstrates the flexibility of the FR starter condensation domains. Finally, expression of the frs genes in E. coli led to heterologous FR production in a cultivable, bacterial host for the first time.

The biocatalytic repertoire of natural biaryl formation.

The catalytic and selective construction of carbon-carbon bonds for the generation of complex molecules is one of the most important tasks in organic chemistry. This was clearly highlighted by the 2010 Nobel Prize in Chemistry, which was awarded for the development of Pd-catalyzed cross-coupling reactions. The underlying concept of cross-linking building blocks to generate molecular complexity can also be widely found in natural product biosynthesis. Impressive examples for such natural cross-coupling reactions are biosynthetic processes for the assembly of biaryl moieties in natural products--highly efficient enzymatic reactions that often achieve synthetically yet unmatched selectivities. This Minireview highlights selected examples that showcase these fascinating biotransformations.