Fungal indole alkaloid biogenesis through evolution of a bifunctional reductase/Diels-Alderase

Abstract

Prenylated indole alkaloids such as the calmodulin-inhibitory malbrancheamides and anthelmintic paraherquamides possess great structural diversity and pharmaceutical utility. Here, we report complete elucidation of the malbrancheamide biosynthetic pathway accomplished through complementary approaches. These include a biomimetic total synthesis to access the natural alkaloid and biosynthetic intermediates in racemic form and in vitro enzymatic reconstitution to provide access to the natural antipode (+)-malbrancheamide. Reductive cleavage of an L-Pro-L-Trp dipeptide from the MalG non-ribosomal peptide synthetase (NRPS) followed by reverse prenylation and a cascade of post-NRPS reactions culminates in an intramolecular [4+2] hetero-Diels-Alder (IMDA) cyclization to furnish the bicyclo[2.2.2]diazaoctane scaffold. Enzymatic assembly of optically pure (+)-premalbrancheamide involves an unexpected zwitterionic intermediate where MalC catalyses enantioselective cycloaddition as a bifunctional NADPH-dependent reductase/Diels-Alderase. The crystal structures of substrate and product complexes together with site-directed mutagenesis and molecular dynamics simulations demonstrate how MalC and PhqE (its homologue from the paraherquamide pathway) catalyse diastereo- and enantioselective cyclization in the construction of this important class of secondary metabolites.

Figure 1.. Fungal bicyclo[2.2.2]diazaoctane indole alkaloids and biosynthesis.

a. Representative natural products with the bicyclo[2.2.2]diazaoctane group colored in red. b. Scheme of malbrancheamide biosynthesis: the MalG NRPS performs reductive offloading of coupled natural substrates l-proline and l-tryptophan to produce the terminal aldehyde 6. This compound undergoes spontaneous cyclization and dehydration to give dienamine 8, which is reverse prenylated by MalE. Compound 9 undergoes spontaneous oxidation to prenylated zwitterion 11. MalC is a bifunctional enzyme which performs reduction and stereoselective [4+2] cycloaddition to furnish (+)-1. The final product malbrancheamide (+)-2 is generated by the MalA halogenase. The products from key biosynthetic steps are colored differently. Proteins are indicated by spheres; MalG domains are adenylation (A₁ and A₂), thiolation (T₁ and T₂), condensation (C) and reductase (R).

Figure 2.. Biomimetic synthesis of premalbrancheamide.

The biomimetic synthesis proceeded through a spontaneous intramolecular [4+2] Diels-Alder reaction from key azadiene intermediate 12 to produce a racemic mixture of syn-premalbrancheamides (1). Zwitterion 11 arises from spontaneous oxidation of 9 and was initially reasoned to be a non-physiological by-product. After discovering that 11 was the preferred substrate of MalC, non-enzymatic chemical reduction by NADH was explored providing 1 in 76% yield from 11. Only optically pure (+)-1 has been isolated from Malbranchea aurantiaca. See SI for complete methods.

Figure 3.. In vitro enzymatic reconstitution of malbrancheamide biosynthesis.

Reactions were monitored by LC/MS. Extracted ion counts (EIC) for key molecules in reaction mixtures are compared to authentic synthetic standards. a. MalG NRPS (excised A₁-T₁, C, T₂, R domains) produced zwitterion 10 by spontaneous oxidation of 8. b – c. Addition of MalE or MalB prenyltransferase formed three products: a prenylated zwitterion 11, and (±)-1. d. MalC Diels-Alderase addition disabled formation of 11 and (−)-1 (see panel f). e. Malbrancheamide 2, the final pathway product, was produced by MalA halogenation of (+)-1. f. Chiral separation of (±)-1 indicates that MalC is an intramolecular [4+2] Diels-Alderase, while neither MalE nor MalB provide enantioselectivity for the spontaneous IMDA reaction. g. MalC-catalyzed reactions under aerobic (11 + MalC) or anaerobic (9 + MalC) conditions. The aerobic route with 11 as the pathway intermediate was more efficient than the anaerobic route from 9. h. Effect of cofactor on the enantiomeric excess of the MalC-catalyzed Diels-Alder reaction. MalC provided limited enantioselectivity when NADH was used as cofactor. EIC traces are colored by compound as in Figure 1b, authentic standards are in purple or pink. For panel g and h, all data represent the average of triplicate independent experiments (center values, mean; error bars, SD; n = 3).

Figure 4.. Structures of MalC and PhqE.

a. MalC tetramer colored by subunit. b. Superposition of MalC and PhqE product complex (gray); NADP⁺ (black C) and premalbrancheamide (green C) are shown as spheres. c. Active site of PhqE·(+)-1·NADP⁺ complex showing close arrangement of the product and the cofactor. d. Omit electron density (F_o-F_c; contoured at 2.2 σ) for the substrate 11 (cyan) in the PhqE/D166N·11·NADP⁺ complex structure. e. Omit electron density (F_o-F_c; contoured at 2.2 σ) for the product 1 (green) in the PhqE·(+)-1·NADP⁺ complex structure. f. Pre-organization for cycloaddition. Substrate 11 binds with the prenyl group poised for the IMDA (dashed lines) in the substrate complex g. Overlay of 11 and premalbrancheamide (+)-1 from the product complex. h. Surface representation of the product complex showing high shape complementarity between premalbrancheamide and PhqE.

Figure 5.. Catalytic mechanism of the MalC/PhqE-catalyzed Diels-Alder reaction.

a – b. Profiles of MalC substrate 11 (blue), MalC product (+)-1 (dark green), and (−)-1 (light green) in the “MalG+MalE+MalC” reconstitution assay. c. MalC product formation assessed by conversion of synthetic 11 to (+)-1. The results agree with those of the reconstitution assay in panel b. Product levels due to non-enzymatic conversion by NADPH were subtracted in all cases. All data represent the average of triplicate independent experiments (center values, mean; error bars, SD; n = 3). d. Proposed catalytic mechanism for MalC/PhqE, with residue numbers for MalC (PhqE residue number = MalC residue number + 1). Arg130 is the indirect proton donor, possibly mediated via the 2’-OH of NADPH ribose. Arg130 forms a salt bridge with Asp108, which is accessible to bulk solvent. Asp165 stabilizes the positive charge of 11, and hydride transfer from NADPH completes the first reduction step, forming an unstable azadiene intermediate. The subsequent IMDA reaction is accelerated primarily via entropy trapping, with diastereo- and enantioselectivity achieved via close packing of the NADP⁺ nicotinamide, the azadiene and MalC Trp168, which together restrain the conformations of both the diene ring and the dienophile to ensure a single cycloaddition mode.