Enzymatic Processing of Fumiquinazoline F: A Tandem Oxidative-Acylation Strategy for the Generation of Multicyclic Scaffolds in Fungal Indole Alkaloid Biosynthesis

Abstract

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) where 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 non-ribosomal peptide synthetase (NRPS) domains through bioinformatics approaches. One domain previously identified is part of the trimodular NRPS Af12080, which we predict is responsible for FQF formation. We now show that two adjacent A. fumigatus ORFs, a monomodular NRPS Af12050 and a flavoprotein Af12060, are necessary and sufficient to convert FQF to FQA. Af12060 oxidizes the 2',3'-double bond of the indole side chain of FQF, and the three-domain NRPS Af12050 activates L-Ala as the adenylate, installs it as the pantetheinyl thioester on its carrier protein domain and acylates the oxidized indole for subsequent intramolecular cyclization to create the 6-5-5 imidazolindolone of FQA. This work provides experimental validation of the fumiquinazoline biosynthetic cluster of A. fumigatus A293, and describes an oxidative annulation biosynthetic strategy likely shared among several classes of polycyclic fungal alkaloids.

Alkaloids from the Sponge‐Associated Fungus Aspergillus sp.

“…[26][27][28] Only one analogous compoung containing a 1-aminocyclopropane-1-carboxylic acid residue, i.e., cottoquinazoline D, has been reported in literature to date. [29] The structurally most similar member and proposed biological precursor of the fumiquinazolines, fumiquinazoline F, is known to be biosynthetically derived from anthranilic acid, tryptophan, and alanine [44] involving a trimodular non-ribosomal peptide synthetase (NRPS). [45] Modification of the indole side chain by oxidative coupling of an additional amino acid molecule, commonly alanine or methylalanine, results in the formation of the imidazoindolone part of the compounds.…”

Nonribosomal Peptide Synthetase Genes pesL and pes1 Are Essential for Fumigaclavine C Production in Aspergillus fumigatus

“…PesL has recently been implicated in fumiquinazoline biosynthesis (1,2), and the observed role for PesL in fumigaclavine C biosynthesis reported here suggests redundant roles for PesL. It remains to be seen whether this is a feature of the remainder of the uncharacterized NRP synthetases within A. fumigatus and other fungi.…”

“…Initially, pesL was proposed to be part of a putative five-gene SM cluster (33) and, more recently, part of an eight-gene cluster proposed to be responsible for the biosynthesis of the fumiquinazoline family of secondary metabolites in A. fumigatus (1). However, coregulated expression of the cluster genes, with or without simultaneous secondary metabolite production, a feature that is a hallmark of SM biosynthetic gene clusters (13,40,47), has not been demonstrated.…”

“…Fumiquinazolines A to G are among a variety of quinazolinecontaining natural products produced by fungi (1,2). Anthranilate (Ant; a nonproteinogenic aryl ␤-amino acid) is a precursor for these compounds (2), whereby a biochemical approach confirmed that an NRP synthetase module (AnaPS module 1) from the known gene cluster for acetylaszonalenin from the Neosartorya fisheri NRRL 181 strain (57) activates Ant.…”

“…The authors of that study proposed that the trimodular NRP synthetase, PesM, is likely to produce fumiquinazoline F, and that an adjacent monomodular NRP synthetase, PesL (AFUA_6G12050), could function in the conversion of fumiquinazoline F to fumiquinazoline A, by activating alanine and acting with an N-acyltransferase (AFUA_6G12100), to couple alanine to both N-1= and C-2= in the indole ring part of fumiquinazoline F (2). Subsequently, recombinantly expressed PesL and an FAD-dependent monooxygenase (AFUA_6G12060) were shown to be required for the conversion of fumiquinazoline F into fumiquinazoline A (1). In contrast to earlier findings about the clustering of genes with pesL (33), the authors noted that pesM is part of an eight-gene cluster, along with pesL, which they predicted to be involved in the production of the Ant-containing alkaloid fumiquinazoline A (1).…”