REVIEW ARTICLE Enzyme evolution in fungal indole alkaloid biosynthesis Amy E. Fraley1,2 and David H. Sherman1,2,3,4 1 Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI, USA 2 Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA 3 Department of Chemistry, University of Michigan, Ann Arbor, MI, USA 4 Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI, USA Keywords The class of fungal indole alkaloids containing the bicyclo[2.2.2]diazaoc- biosynthesis; Diels–Alderase; natural tane ring is comprised of diverse molecules that display a range of biologi- products; nonribosomal peptides; cal activities. While much interest has been garnered due to their monooxygenase therapeutic potential, this class of molecules also displays unique chemical Correspondence functionality, making them intriguing synthetic targets. Many elegant and D. H. Sherman, Life Sciences Institute, 210 intricate total syntheses have been developed to generate these alkaloids, Washtenaw Avenue, Ann Arbor, MI 48104, but the selectivity required to produce them in high yield presents great USA barriers. Alternatively, if we can understand the molecular mechanisms Tel: +734 615 9907 behind how fungi make these complex molecules, we can leverage the E-mail: [email protected] power of nature to perform these chemical transformations. Here, we describe the various studies regarding the evolutionary development of (Received 21 August 2019, revised 24 November 2019, accepted 27 February enzymes involved in fungal indole alkaloid biosynthesis. 2020) doi:10.1111/febs.15270 Introduction to fungal indole alkaloids The fungal indole alkaloid class of natural products knowledge gaps with detailed biochemical characteri- contains molecules with unique structural properties zation. Multiple strategies including molecular and and a variety of biological activities. In particular, structural biology have brought our foundational the subgroup typified by the bicyclo[2.2.2]diazaoctane understanding of these pathways to the forefront of ring has been extensively studied within our group. the field. Initially, feeding studies using isotopically labeled pre- Our work has focused on the families of monoke- cursors and biomimetic chemical syntheses provided topiperazines (MKPs) such as the paraherquamides insight into the biosynthetic pathways for these mole- (Phq) [2,3] and malbrancheamides (Mal) [4], and dike- cules. These efforts were later supplemented with gen- topiperazines (DKP) such as the stephacidins [5], ome sequencing and bioinformatic analyses [1], notoamides (Not) [6-8], and brevianamides (Bvn) greatly advancing our understanding of how these (Figs 1–3) [9]. These molecules display significant bio- natural products are generated, yet we have only logical activities with potential therapeutic value such recently utilized our capabilities to address the as anthelmintics [10-13], vasodilators [14], and Abbreviations A, adenylation domain; Asn, asparagine,; C, condensation domain; DKP, diketopiperazine; FAD, flavin adenine dinucleotide; FDH, flavin- dependent halogenase; FMO, flavin-dependent monooxygenase; IMDA, intramolecular Diels–Alder; Lys, lysine; MKP, monoketopiperazine; NADPH, nicotinamide adenine dinucleotide phosphate; NRPS, nonribosomal peptide synthetase; Pro, proline; R, reductase domain; SDR, short-chain dehydrogenase; Ser, serine; T, thiolation domain; Trp, tryptophan; Tyr, tyrosine. The FEBS Journal 287 (2020) 1381–1402 ª 2020 Federation of European Biochemical Societies 1381 Enzyme evolution in fungal indole alkaloid biosynthesis A. E. Fraley and D. H. Sherman A T C A T R O O S O S O MalG HN HN NADPH -H2O HN N NH NH2 O OH H N NH NH NH MalC O H NADP+ N N O O OH Premalbrancheamide (23) HN MalE HN N H DMAPP N Cl N N N MalA FADH2 O H N NH NH NH N Malbrancheamide B (68) 6 74 MalC 22 H Spontaneous Oxidation NADPH N O O MalE H Cl N N N O H DMAPP N N N N HO N Isomalbrancheamide B (69) H N N NH NH MalA Cl FADH2 75 21 O H Cl N N Malbrancheamide (67) Fig. 1. Malbrancheamide biosynthetic pathway. The pathway includes nonribosomal peptide synthetase (NRPS) MalG, prenyltransferase MalE, intramolecular Diels–Alderase MalC, and flavin-dependent halogenase MalA. MalG and MalC require nicotinamide adenine dinucleotide phosphate (NADPH) cofactor for catalysis, while MalA requires the reduced flavin adenine dinucleotide (FADH2) cofactor provided by a reductase partner. MalE prenylates the indole C2 position with a prenyl group provided by dimethylallyl pyrophosphate (DMAPP). anticancer agents [15]. All bear a similar core scaffold, Distinct nonribosomal peptide but vary structurally based on key functionalities synthetases including oxidation, halogenation, and additional ring systems. Thus, our initial bioinformatic analyses have The first step in the biosynthesis of these fungal indole enabled us to link the construction of the common alkaloids is catalyzed by the multidomain nonriboso- core to the homologous enzymes, while cluster-specific mal peptide synthetases (NRPSs). The architecture of genes provide the basis for structural differences [1]. the NRPS consists of adenylation (A), thiolation (T), There is a clear evolutionary divergence between the condensation (C), and reductase (R) domains, where MKP and DKP containing families within this class. the first A domain in this class of compounds is selec- Thus, the installation of similar functional groups may tive for proline, distinguishing these systems from be accomplished by different types of enzymes. Addi- other fungal bimodular NRPS pathways. The domain tionally, parallel work in pathways that do not contain construction of the terminal module determines the the core bicycle reveals compelling biosynthetic branch oxidation state of the offloaded dipeptide, and subse- points. Extensive characterization of the fumitremorgin quently the core scaffold. The DKP containing mole- [16,17], spirotryprostatin, [18] and aurachin [19,20] cules are produced by an NRPS with A-T-C-A-T-C families by a number of groups has provided insights domain organization, whereas the MKP containing into the homologous transformations within these var- molecules involve an NRPS with A-T-C-A-T-R orga- ied bicyclo[2.2.2]diazaoctane ring-containing metabolic nization. For example, the notoamide (DKP) biosyn- systems. thetic pathway contains an NRPS with a terminal 1382 The FEBS Journal 287 (2020) 1381–1402 ª 2020 Federation of European Biochemical Societies A. E. Fraley and D. H. Sherman Enzyme evolution in fungal indole alkaloid biosynthesis A T C A T R O O OH S O S O PhqB PhqI NADPH HN DMAPP HN N N N N NH NH2 NH NH NH NH PhqE PhqE 776NADPH 78 NADP+ Spontaneous Oxidation O O PhqI H N DMAPP N N N N HO N N NH NH 77 24 H H Divergence Between N O N O Pyran and Dioxepin Metabolites O O H O H H N N N N N PhqK PT, [ox] Paraherquamide K (26) FAD, NADH O H N N O O H H Preparaherquamide (25) N O N O O O H O H N N N N Semi-Pinacol Rearrangement Paraherquamide L (27) O O O NH NH NH OH O H O O N O N O N O N N N Paraherquamide M (79) Paraherquamide F (81) Paraherquamide G (83) [ox] O N-MT O O NH NH NH O O O OH O H O O N O N O N O N N N Paraherquamide N (80) Paraherquamide E (82) Paraherquamide A (1) Fig. 2. Paraherquamide biosynthetic pathway. The pathway includes nonribosomal peptide synthetase (NRPS) PhqB, prenyltransferase PhqI, intramolecular Diels–Alderase PhqE, and flavin-dependent monooxygenase PhqK. A putative prenyltransferase and oxidative enzyme are proposed to be involved in the formation of the pyran and dioxepin rings. PhqB and PhqE require nicotinamide adenine dinucleotide phosphate (NADPH) cofactor for catalysis, while PhqK requires flavin adenine dinucleotide (FAD) and the reduced nicotinamide adenine dinucleotide (NADH) cofactor. PhqI prenylates the indole C2 position with a prenyl group provided by dimethylallyl pyrophosphate (DMAPP). condensation domain (NotE) [21], while the mal- reductase domains [1,22]. The different terminal brancheamide and paraherquamide (MKPs) biosyn- domains and the coinciding mono- or diketopiperazine thetic pathways include NRPSs with terminal systems indicate that the NRPSs offload distinct The FEBS Journal 287 (2020) 1381–1402 ª 2020 Federation of European Biochemical Societies 1383 Enzyme evolution in fungal indole alkaloid biosynthesis A. E. Fraley and D. H. Sherman A T C A T C O O O Reverse Normal S S O O HN Prenylation HN HN Prenylation N N N NotE/NotE' NotF/NotF' NotC/NotC' NH NH2 NADPH O DMAPP O NotG/NotG' O DMAPP N N N 3 HO N H H H 2 H 1 Brevianamide F (4) Deoxybrevianamide E (16) 6-OH-deoxybrevianamide E (84) O O O HN HN O HN N N N HO NotD/ NotB/NotB' N O NotD' ? O FAD, NADPH N O O N HO O N O N O and O N 1 H H Brevianamide A- H H 2 3 Notoamide S (5) Notoamide E (15) Notoamide D (20) Notoamide C (19) like route? NotH/NotH' ? NotH/NotH' ? 2 e- [ox] 2 e- [ox] IMDA IMDA H H O O OH N OH N O NH N NotD/ 6 NotI/NotI' NotD' ? FAD, NADH O H O H O H O H N N N O N O N N N N O O O O (+)-Notoamide T (28) (+)-Stephacidin A (10) (-)-Notoamide B (12) (-)-Notoamide A (86) A. protuberus A. protuberus H H O HO O HO N O N HN N 6 H O H O H O H O N N O N O N N N N N O O O O (-)-Notoamide T (29) (-)-Stephacidin A (13) (+)-Notoamide B (14) (+)-Notoamide A (87) A. amoenus A. amoenus H H O N OH N O NH 6 O H O H O H O N N N N N N O O O (+)-6-epi-Notoamide T (33) (+)-6-epi-Stephacidin A (9) (+)-Versicolamide B (8) A.