Fungal Indole Alkaloid Biogenesis Through Evolution of a Bifunctional Reductase/Diels-Alderase

Abstract

Prenylated indole alkaloids isolated from various fungi possess great structural diversity and pharmaceutical utility. Among them are the calmodulin inhibitory malbrancheamides and paraherquamides, used as anthelmintics in animal health. Herein, 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 that provides access to the natural antipode (+)-malbrancheamide. Reductive cleavage of a L-Pro-L-Trp dipeptide from the MalG nonribosomal 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 catalyzes enantioselective cycloaddition as a bifunctional NADPH-dependent reductase/Diels-Alderase. Crystal structures of substrate and product complexes together with site-directed mutagenesis and molecular dynamics simulations demonstrated how MalC and PhqE, its homolog from the paraherquamide pathway, catalyze diastereo- and enantioselective cyclization in the construction of this important class of secondary metabolites.

Enzyme evolution in fungal indole alkaloid biosynthesis

“…The DKP containing molecules are produced by an NRPS with A‐T‐C‐A‐T‐C domain organization, whereas the MKP containing molecules involve an NRPS with A‐T‐C‐A‐T‐R organization. For example, the notoamide (DKP) biosynthetic pathway contains an NRPS with a terminal condensation domain (NotE) [21], while the malbrancheamide and paraherquamide (MKPs) biosynthetic pathways include NRPSs with terminal reductase domains [1,22]. The different terminal domains and the coinciding mono‐ or diketopiperazine systems indicate that the NRPSs offload distinct dipeptide intermediates for the subsequent intramolecular Diels–Alder (IMDA) reaction.…”

“…The mechanism for the MKP‐producing NRPS has been identified based on recent investigations of the Phq and Mal systems by our group [22]. The distinguishing terminal reductive domain belongs to the family of short‐chain dehydrogenase reductase (SDR) nicotinamide adenine dinucleotide phosphate (NAD(P)H)‐dependent oxidoreductases.…”

“…The NRPS couples l ‐proline and l ‐tryptophan to produce an l ‐Pro‐ l ‐Trp aldehyde product through cyclization and dehydration (Figs 1 and 2). Under conditions that contain the NRPS alone, the linear aldehyde product spontaneously oxidizes to a cyclic aromatic zwitterionic species [22]. This catalytic mechanism requires the NADPH cofactor to provide the reducing equivalents, and both PhqB and MalG R‐domains contain the canonical SDR tyrosine and lysine active site residues (Fig.…”

“…This catalytic mechanism requires the NADPH cofactor to provide the reducing equivalents, and both PhqB and MalG R‐domains contain the canonical SDR tyrosine and lysine active site residues (Fig. 4) [22].…”

“…We have found that most of the bicyclo[2.2.2]diazaoctane fungal indole alkaloid gene clusters contain an additional prenyltransferase relative to the number of prenyl units incorporated into the molecule. The initial reverse prenylation occurs at the indole C2 position of the NRPS dipeptide product[22], while prenylation of the phenolic portion of the indole to form the pyran and dioxepin rings can occur before (Fig. 3) or after (Fig.…”