Product Selectivity in Baeyer-Villiger Monooxygenase-Catalyzed Bacterial Alkaloid Core Structure Maturation.

Baeyer-Villiger monooxygenases (BVMOs) play crucial roles in the core-structure modification of natural products. They catalyze lactone formation by selective oxygen insertion into a carbon-carbon bond adjacent to a carbonyl group (Baeyer-Villiger oxidation, BVO). The homologous bacterial BVMOs, BraC and PxaB, thereby process bicyclic dihydroindolizinone substrates originating from a bimodular nonribosomal peptide synthetase (BraB or PxaA). While both enzymes initially catalyze the formation of oxazepine-dione intermediates following the identical mechanism, the final natural product spectrum diverges. For the pathway involving BraC, the exclusive formation of lipocyclocarbamates, the brabantamides, was reported. The pathway utilizing PxaB solely produces pyrrolizidine alkaloids, the pyrrolizixenamides. Surprisingly, replacing pxaB within the pyrrolizixenamide biosynthetic pathway by braC does not change the product spectrum to brabantamides. Factors controlling this product selectivity have remained elusive. In this study, we set out to solve this puzzle by combining the total synthesis of crucial pathway intermediates and anticipated products with in-depth functional in vitro studies on both recombinant BVMOs. This work shows that the joint oxazepine-dione intermediate initially formed by both BVMOs leads to pyrrolizixenamides upon nonenzymatic hydrolysis, decarboxylative ring contraction, and dehydration. Brabantamide biosynthesis is enzyme-controlled, with BraC efficiently transforming all the accepted substrates into its cognate final product scaffold. PxaB, in contrast, shows only considerable activity toward brabantamide formation for the substrate analog with a natural brabantamide-type side chain structure, revealing substrate-controlled product selectivity.

Discovery of extended product structural space of the fungal dioxygenase AsqJ.

The fungal dioxygenase AsqJ catalyses the conversion of benzo[1,4]diazepine-2,5-diones into quinolone antibiotics. A second, alternative reaction pathway leads to a different biomedically important product class, the quinazolinones. Within this work, we explore the catalytic promiscuity of AsqJ by screening its activity across a broad range of functionalized substrates made accessible by solid-/liquid-phase peptide synthetic routes. These systematic investigations map the substrate tolerance of AsqJ within its two established pathways, revealing significant promiscuity, especially in the quinolone pathway. Most importantly, two further reactivities leading to new AsqJ product classes are discovered, thus significantly expanding the structural space accessible by this biosynthetic enzyme. Switching AsqJ product selectivity is achieved by subtle structural changes on the substrate, revealing a remarkable substrate-controlled product selectivity in enzyme catalysis. Our work paves the way for the biocatalytic synthesis of diverse biomedically important heterocyclic structural frameworks.

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.

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.

Fungal Dioxygenase AsqJ Is Promiscuous and Bimodal: Substrate-Directed Formation of Quinolones versus Quinazolinones.

Previous studies showed that the FeII /α-ketoglutarate dependent dioxygenase AsqJ induces a skeletal rearrangement in viridicatin biosynthesis in Aspergillus nidulans, generating a quinolone scaffold from benzo[1,4]diazepine-2,5-dione substrates. We report that AsqJ catalyzes an additional, entirely different reaction, simply by a change in substituent in the benzodiazepinedione substrate. This new mechanism is established by substrate screening, application of functional probes, and computational analysis. AsqJ excises H2 CO from the heterocyclic ring structure of suitable benzo[1,4]diazepine-2,5-dione substrates to generate quinazolinones. This novel AsqJ catalysis pathway is governed by a single substituent within the complex substrate. This unique substrate-directed reactivity of AsqJ enables the targeted biocatalytic generation of either quinolones or quinazolinones, two alkaloid frameworks of exceptional biomedical relevance.

Bypassing Biocatalytic Substrate Limitations in Oxidative Dearomatization Reactions by Transient Substrate Mimicking.

Enzymatic oxidative dearomatization is an efficient way to generate chiral molecules from simple arenes. One example is the flavin-dependent monooxygenase SorbC involved in sorbicillinoid biosynthesis. However, SorbC requires a long-chain keto substituent at its phenolic substrate, thus preventing its application beyond the synthesis of natural sorbicillinoids or close structural analogues. This work describes an approach to broaden the accessible product spectrum of SorbC by employing an ester functionality mimicking the natural substrate structure during enzymatic oxidation.