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) 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. 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 Af293 and describes an oxidative annulation biosynthetic strategy likely shared among several classes of polycyclic fungal alkaloids.

Figure 1

(A) Fumiquinazoline gene cluster and (B) proposed biosynthetic route to fumiquinazoline A in Aspergillus fumigatus Af293.

Figure 2

Examples of fungal metabolites containing a multicyclic indolic scaffold likely derived in part or in whole from an oxidative-coupling transformation of indole with an additional amino acid in a manner similar to the generation of the imidazoindolone moiety of fumiquinazoline A. The indolic C2' and C3' positions are labeled for clarity.

Figure 3

Biochemical characterization and proposed mechanism of the FAD and NAD(P)H-dependent monooxygenase, Af12060. (A) HPLC analysis (274_nm detection) of reactions containing 200 μM FQF, 5 μM Af12060, and ± 1 mM NADPH or NADH. Reactions were quenched after 40 min incubation at 25°C by adding 50% MeCN. (B) Time course for FQF utilization and accompanying rate data obtained by integration of FQF peak area. Reaction setup used 200 μM FQF, 2 μM Af12060, and 1 mM NADPH; reactions were quenched at the indicated timepoint by addition of MeCN. (C) Proposed pathway for the conversion of FQF to 2 and 3 by Af12060. Brackets denote putative intermediates not detected by HPLC or LC-MS analysis. Possible structures for the oxidized dimer species (4) are provided in Figure S9A.

Figure 4

Characterization of substrate-dependent adenylation activity and L-Ala loading of Af12050. (A) Schematic for the two-step process of adenylation (to generate an activated L-alanyl-AMP intermediate) and subsequent loading of the L-alanyl group onto the 4'-phosphopantatheine group of holo-protein. (B) ATP-[³²P]PP_i exchange activity promoted by apo-Af12050 with various amino acids tested as substrates (100% activity corresponds to 112,000 CPM). Reactions were quenched with activated charcoal following 1.5 hr incubation at 25°C and radioactivity detected by liquid scintillation counting. (C) Time course for the loading of radiolabeled [¹⁴C]L-Ala onto Af12050 protein which was purified directly from E. coli BL21(DE3) cells (filled circles, apo-protein), or following in vitro incubation with B. subtilus Sfp and CoA for T-domain phosphopantetheinylation (filled squares, holo-protein).

Figure 5

Reconstitution of fumiquinazoline A (FQA) biosynthesis by combining Af12060 (monooxygenase) and Af12050 (non-ribosomal peptide synthetase) with FQF as substrate. (A) HPLC analysis of reactions containing 200 μM FQF, 1 mM NADPH, 1 mM L-Ala, 2 mM Mg²⁺, and various combinations of 2.5 μM Af12060 (abbreviated as “60”), 5 μM Af12050 (abbreviated as “50”), and 1 mM ATP. The (-) enzyme control contains all reaction components except Af12050 and Af12060. Holo-50 denotes phosphopantetheinylated protein. (B) Time course of FQA production and accompanying rate data for FQF utilization and FQA formation. The concentrations of components used were the same as for (A) except for that 1 μM Af12060 was used. (C) Two proposed pathways for the conversion of FQF to FQA by Af12060 and Af12050. Brackets denote putative intermediates not detected by HPLC or LC-MS analysis. The C-domain of Af12050 is postulated to catalyze the two step process of L-Ala coupling and subsequent intramolecular attack to generate the 6-5-5 imidazoindolone moiety of FQA.