De novo design of proteins housing excitonically coupled chlorophyll special pairs.

Natural photosystems couple light harvesting to charge separation using a 'special pair' of chlorophyll molecules that accepts excitation energy from the antenna and initiates an electron-transfer cascade. To investigate the photophysics of special pairs independently of the complexities of native photosynthetic proteins, and as a first step toward creating synthetic photosystems for new energy conversion technologies, we designed C-symmetric proteins that hold two chlorophyll molecules in closely juxtaposed arrangements. X-ray crystallography confirmed that one designed protein binds two chlorophylls in the same orientation as native special pairs, whereas a second designed protein positions them in a previously unseen geometry.

Mechanism of the P450-Catalyzed Oxidative Cyclization in the Biosynthesis of Griseofulvin.

Griseofulvin is an anti-fungal agent which has recently been determined to have potential anti-viral and anti-cancer applications. The role of specific enzymes involved in the biosynthesis of this natural product has previously been determined, but the mechanism by which a p450, GsfF, catalyzes the key oxidative cyclization of griseophenone B remains unknown. Using density functional theory (DFT), we have determined the mechanism of this oxidation that forms the oxa-spiro core of griseofulvin.

Structural basis of nonribosomal peptide macrocyclization in fungi.

Nonribosomal peptide synthetases (NRPSs) in fungi biosynthesize important pharmaceutical compounds, including penicillin, cyclosporine and echinocandin. To understand the fungal strategy of forging the macrocyclic peptide linkage, we determined the crystal structures of the terminal condensation-like (C) domain and the holo thiolation (T)-C complex of Penicillium aethiopicum TqaA. The first, to our knowledge, structural depiction of the terminal module in a fungal NRPS provides a molecular blueprint for generating new macrocyclic peptide natural products.

Reconstitution of Fungal Nonribosomal Peptide Synthetases in Yeast and In Vitro.

The emergence of next-generation sequencing has provided new opportunities in the discovery of new nonribosomal peptides (NRPs) and NRP synthethases (NRPSs). However, there remain challenges for the characterization of these megasynthases. While genetic methods in native hosts are critical in elucidation of the function of fungal NRPS, in vitro assays of intact heterologously expressed proteins provide deeper mechanistic insights in NRPS enzymology.

Understanding Programming of Fungal Iterative Polyketide Synthases: The Biochemical Basis for Regioselectivity by the Methyltransferase Domain in the Lovastatin Megasynthase.

Highly reducing polyketide synthases (HR-PKSs) from fungi synthesize complex natural products using a single set of domains in a highly programmed, iterative fashion. The most enigmatic feature of HR-PKSs is how tailoring domains function selectively during different iterations of chain elongation to afford structural diversity. Using the lovastatin nonaketide synthase LovB as a model system and a variety of acyl substrates, we characterized the substrate specificity of the LovB methyltransferase (MT) domain.

Discovery of Unclustered Fungal Indole Diterpene Biosynthetic Pathways through Combinatorial Pathway Reassembly in Engineered Yeast.

The structural diversity and biological activities of fungal indole diterpenes (IDTs) are generated in large part by the IDT cyclases (IDTCs). Identifying different IDTCs from IDT biosynthetic pathways is therefore important toward understanding how these enzymes introduce chemical diversity from a common linear precursor. However, IDTCs involved in the cyclization of the well-known aflavinine subgroup of IDTs have not been discovered.

Tandem prenyltransferases catalyze isoprenoid elongation and complexity generation in biosynthesis of quinolone alkaloids.

Modification of natural products with prenyl groups and the ensuing oxidative transformations are important for introducing structural complexity and biological activities. Penigequinolones (1) are potent insecticidal alkaloids that contain a highly modified 10-carbon prenyl group. Here we reveal an iterative prenylation mechanism for installing the 10-carbon unit using two aromatic prenyltransferases (PenI and PenG) present in the gene cluster of 1 from Penicillium thymicola.

Next-generation sequencing approach for connecting secondary metabolites to biosynthetic gene clusters in fungi.

Genomics has revolutionized the research on fungal secondary metabolite (SM) biosynthesis. To elucidate the molecular and enzymatic mechanisms underlying the biosynthesis of a specific SM compound, the important first step is often to find the genes that responsible for its synthesis. The accessibility to fungal genome sequences allows the bypass of the cumbersome traditional library construction and screening approach.

A cytochrome P450 serves as an unexpected terpene cyclase during fungal meroterpenoid biosynthesis.

Viridicatumtoxin (1) is a tetracycline-like fungal meroterpenoid with a unique, fused spirobicyclic ring system. Puzzlingly, no dedicated terpene cyclase is found in the gene cluster identified in Penicillium aethiopicum. Cytochrome P450 enzymes VrtE and VrtK in the vrt gene cluster were shown to catalyze C5-hydroxylation and spirobicyclic ring formation, respectively.

Complexity generation in fungal polyketide biosynthesis: a spirocycle-forming P450 in the concise pathway to the antifungal drug griseofulvin.

Griseofulvin (1) is a spirocyclic fungal natural product used in treatment of fungal dermatophytes. Formation of the spirocycle, or the grisan scaffold, from a benzophenone precursor is critical for the activity of 1. In this study, we have systematically characterized each of the biosynthetic enzymes related to the biogenesis of 1, including the characterization of a new polyketide synthase GsfA that synthesizes the benzophenone precursor and a cytochrome P450 GsfF that performs oxidative coupling between the orcinol and the phloroglucinol rings to yield the grisan structure.

Discovery of cryptic polyketide metabolites from dermatophytes using heterologous expression in Aspergillus nidulans.

Dermatophytes belonging to the Trichophyton and Arthroderma genera cause skin infections in humans and animals. From genome sequencing data, we mined a conserved gene cluster among dermatophytes that are homologous to one that produces an immunosuppressive polyketide in Aspergillus fumigatus. Using a recombination-based cloning strategy in yeast, we constructed fungal heterologous expression vectors that encode the cryptic clusters.

EcdGHK are three tailoring iron oxygenases for amino acid building blocks of the echinocandin scaffold.

The echinocandins are a small group of fungal N-acylated cyclic hexapeptides that are fungicidal for candida strains and fungistatic for aspergilli by targeting cell wall 1,3-β-glucan synthases. The side chains of all six amino acid building blocks have hydroxyl groups, including the nonproteinogenic 4R,5R-dihydroxy-Orn1, 4R-OH-Pro3, 3S,4S-dihydroxy-homoTyr4, and 3S-OH-4S-Me-Pro6. The echinocandin (ecd) gene cluster contains two predicted nonheme mononuclear iron oxygenase genes (ecdG,K) and one encoding a P450 type heme protein (ecdH).

Bringing protein engineering and natural product biosynthesis together.

In this issue of Chemistry & Biology, Zhang and colleagues developed a yeast cell surface display strategy to effectively evolve the substrate specificity of DhbE, one of the adenylation domains of the bacillibactin synthetase complex. The method yields DbhE variants that have dramatically altered substrate specifities toward unnatural aryl substrates.

Identification and characterization of the echinocandin B biosynthetic gene cluster from Emericella rugulosa NRRL 11440.

Echinocandins are a family of fungal lipidated cyclic hexapeptide natural products. Due to their effectiveness as antifungal agents, three semisynthetic derivatives have been developed and approved for treatment of human invasive candidiasis. All six of the amino acid residues are hydroxylated, including 4R,5R-dihydroxy-L-ornithine, 4R-hydroxyl-L-proline, 3S,4S-dihydroxy-L-homotyrosine, and 3S-hydroxyl-4S-methyl-L-proline.

Protein engineering towards natural product synthesis and diversification.

A dazzling array of enzymes is used by nature in making structurally complex natural products. These enzymes constitute a molecular toolbox that may be used in the construction and fine-tuning of pharmaceutically active molecules. Aided by technological advancements in protein engineering, it is now possible to tailor the activities and specificities of these enzymes as biocatalysts in the production of both natural products and their unnatural derivatives.