Short pathways to complexity generation: fungal peptidyl alkaloid multicyclic scaffolds from anthranilate building blocks.

Complexity generation in naturally occurring peptide scaffolds can occur either by posttranslational modifications of nascent ribosomal proteins or through post assembly line tailoring of nonribosomal peptides. Short enzymatic pathways utilizing bimodular and trimodular nonribosomal peptide synthetase (NRPS) assembly lines, followed by tailoring oxygenases and/or prenyltransferases, efficiently construct complex fungal peptidyl alkaloid scaffolds in Aspergilli, Neosartorya, and Penicillium species. Use of the nonproteinogenic amino acid anthranilate as chain-initiating building block and chain-terminating intramolecular nucleophile leads efficiently to peptidyl alkaloid scaffolds with two to seven fused rings.

Cyclization of fungal nonribosomal peptides by a terminal condensation-like domain.

Cyclization of linear peptidyl precursors produced by nonribosomal peptide synthetases (NRPSs) is an important step in the biosynthesis of bioactive cyclic peptides. Whereas bacterial NRPSs use thioesterase domains to perform the cyclization, fungal NRPSs have apparently evolved to use a different enzymatic route. In verified fungal NRPSs that produce macrocyclic peptides, each megasynthetase terminates with a condensation-like (C(T)) domain that may perform the macrocyclization reaction.

Crystal structure and biochemical studies of the trans-acting polyketide enoyl reductase LovC from lovastatin biosynthesis.

Lovastatin is an important statin prescribed for the treatment and prevention of cardiovascular diseases. Biosynthesis of lovastatin uses an iterative type I polyketide synthase (PKS). LovC is a trans-acting enoyl reductase (ER) that specifically reduces three out of eight possible polyketide intermediates during lovastatin biosynthesis.

Insight into the molecular basis of aromatic polyketide cyclization: crystal structure and in vitro characterization of WhiE-ORFVI.

Aromatic polyketides are biologically active natural products. Many important pharmaceuticals are derived from aromatic polyketides. Especially important in aromatic polyketide biosynthesis is the regiospecific cyclization of a linear, preassembled polyketide chain catalyzed by aromatase/cyclase (ARO/CYC), which serves as a key control point in aromatic ring formation.

Aminobenzoates as building blocks for natural product assembly lines.

The ortho-, meta-, and para- regioisomers of aminobenzoate are building blocks for a wide range of microbial natural products. Both the ortho-isomer (anthranilate) and PABA derive from the central shikimate pathway metabolite chorismate while the meta-isomer is not available by that route and starts from UDP-3-aminoglucose. PABA is largely funnelled into folate biosynthesis while anthranilate is the scaffold for biosynthetic elaboration into many natural heterocycles, most notably with its role in indole formation for tryptophan biosynthesis.

Complexity generation in fungal peptidyl alkaloid biosynthesis: oxidation of fumiquinazoline A to the heptacyclic hemiaminal fumiquinazoline C by the flavoenzyme Af12070 from Aspergillus fumigatus.

The human pathogen Aspergillus fumigatus makes a series of fumiquinazoline (FQ) peptidyl alkaloids of increasing scaffold complexity using L-Trp, 2 equiv of L-Ala, and the non-proteinogenic amino acid anthranilate as building blocks. The FQ gene cluster encodes two non-ribosomal peptide synthetases (NRPS) and two flavoproteins. The trimodular NRPS Af12080 assembles FQF (the first level of complexity) while the next two enzymes, Af12060 and Af12050, act in tandem in an oxidative annulation sequence to couple alanine to the indole side chain of FQF to yield the imidazolindolone-containing FQA.

Structural and biochemical characterization of ZhuI aromatase/cyclase from the R1128 polyketide pathway.

Aromatic polyketides comprise an important class of natural products that possess a wide range of biological activities. The cyclization of the polyketide chain is a critical control point in the biosynthesis of aromatic polyketides. The aromatase/cyclases (ARO/CYCs) are an important component of the type II polyketide synthase (PKS) and help fold the polyketide for regiospecific cyclizations of the first ring and/or aromatization, promoting two commonly observed first-ring cyclization patterns for the bacterial type II PKSs: C7-C12 and C9-C14.

Identification of phenylalanine 3-hydroxylase for meta-tyrosine biosynthesis.

Phenylalanine hydroxylase (PheH) is an iron(II)-dependent enzyme that catalyzes the hydroxylation of aromatic amino acid l-phenylalanine (L-Phe) to l-tyrosine (L-Tyr). The enzymatic modification has been demonstrated to be highly regiospecific, forming proteinogenic para-Tyr (p-Tyr) exclusively. Here we biochemically characterized the first example of a phenylalanine 3-hydroxylase (Phe3H) that catalyzes the synthesis of meta-Tyr (m-Tyr) from Phe.

Unraveling terminal C-domain-mediated condensation in fungal biosynthesis of imidazoindolone metabolites.

The fungal peptidyl alkaloids of the tryptoquialanine and fumiquinazoline families are nonribosomally assembled by annulation of the indole side chain of fumiquinazoline F (FQF) with an alaninyl or aminoisobutyryl unit by monomodular NRPS enzymes containing adenylation, thiolation, and condensation (A-T-C) domains. The Af12060 and Af12050 enzyme pair from Aspergillus fumigatus thereby converts FQF to FQA, while the homologous TqaH and TqaB enzyme pair from Penicillium aethiopicum makes the 2'-epi diastereomer of FQA, differing only in the stereochemistry of one of the C-N bonds formed in the annulation with l-Ala. To evaluate the basis for this stereochemical control, we have mixed and matched the flavoprotein oxygenases Af12060 and TqaH with the A-T-C modular enzymes Af12050 and TqaB to show that the NRPS enzymes control the stereochemical outcome.

Fungal indole alkaloid biosynthesis: genetic and biochemical investigation of the tryptoquialanine pathway in Penicillium aethiopicum.

Tremorgenic mycotoxins are a group of indole alkaloids which include the quinazoline-containing tryptoquivaline (2) that are capable of eliciting intermittent or sustained tremors in vertebrate animals. The biosynthesis of this group of bioactive compounds, which are characterized by an acetylated quinazoline ring connected to a 6-5-5 imidazoindolone ring system via a 5-membered spirolactone, has remained uncharacterized. Here, we report the identification of a gene cluster (tqa) from P.

Enzymatic processing of fumiquinazoline F: a tandem oxidative-acylation strategy for the generation of multicyclic scaffolds in fungal indole alkaloid biosynthesis.

Aspergillus fumigatus Af293 is a known producer of quinazoline natural products, including the antitumor fumiquinazolines, of which the simplest member is fumiquinazoline F (FQF) with a 6-6-6 tricyclic core derived from anthranilic acid, tryptophan, and alanine. FQF is the proposed biological precursor to fumiquinazoline A (FQA) in which the pendant indole side chain has been modified via oxidative coupling of an additional molecule of alanine, yielding a fused 6-5-5 imidazoindolone. We recently identified fungal anthranilate-activating nonribosomal peptide synthetase (NRPS) domains through bioinformatics approaches.

Anthranilate-activating modules from fungal nonribosomal peptide assembly lines.

Fungal natural products containing benzodiazepinone- and quinazolinone-fused ring systems can be assembled by nonribosomal peptide synthetases (NRPS) using the conformationally restricted beta-amino acid anthranilate as one of the key building blocks. We validated that the first module of the acetylaszonalenin synthetase of Neosartorya fischeri NRRL 181 activates anthranilate to anthranilyl-AMP. With this as a starting point, we then used bioinformatic predictions about fungal adenylation domain selectivities to identify and confirm an anthranilate-activating module in the fumiquinazoline A producer Aspergillus fumigatus Af293 as well as a second anthranilate-activating NRPS in N.

Structural enzymology of polyketide synthases.

This chapter describes structural and associated enzymological studies of polyketide synthases, including isolated single domains and multidomain fragments. The sequence-structure-function relationship of polyketide biosynthesis, compared with homologous fatty acid synthesis, is discussed in detail. Structural enzymology sheds light on sequence and structural motifs that are important for the precise timing, substrate recognition, enzyme catalysis, and protein-protein interactions leading to the extraordinary structural diversity of naturally occurring polyketides.

Crystal structure and functional analysis of tetracenomycin ARO/CYC: implications for cyclization specificity of aromatic polyketides.

Polyketides are a class of natural products with highly diverse chemical structures and pharmaceutical activities. Polyketide cyclization, promoted by the aromatase/cyclase (ARO/CYC), helps diversify aromatic polyketides. How the ARO/CYC promotes highly specific cyclization is not well understood because of the lack of a first-ring ARO/CYC structure.

Engineered biosynthesis of a novel amidated polyketide, using the malonamyl-specific initiation module from the oxytetracycline polyketide synthase.

Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases (PKSs). Understanding the biochemistry of tetracycline PKSs is an important step toward the rational and combinatorial manipulation of tetracycline biosynthesis. To this end, we have sequenced the gene cluster of oxytetracycline (oxy and otc genes) PKS genes from Streptomyces rimosus.