Complexity and Diversity Generation in the Biosynthesis of

Feb 14, 2019 - Here, we characterized the use of an α-KG/Fe(II)-dependent dioxygenase (α-KGD) as a new strategy in Nature to increase structural com...
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Complexity and Diversity Generation in the Biosynthesis of Fumiquinazoline-Related Peptidyl Alkaloids Daojiang Yan,†,§ Qibin Chen,†,§ Jie Gao,†,§ Jian Bai,† Bingyu Liu,† Yalong Zhang,† Le Zhang,† Chen Zhang,† Yi Zou,‡ and Youcai Hu*,† †

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, P.R. China ‡ College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, P.R. China

Org. Lett. Downloaded from pubs.acs.org by TULANE UNIV on 02/14/19. For personal use only.

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ABSTRACT: Fumiquinazolines are multicyclic peptidyl alkaloids where FAD-dependent oxidases are main tailing redox enzymes in their biosynthesis. Here, we characterized the use of an α-KG/Fe(II)-dependent dioxygenase (α-KGD) as a new strategy in Nature to increase structural complexity in fumiquinazolines biosynthesis by elucidating the concise three enzymes biosynthetic pathway of heptacyclical alanditrypinone (1). Further genome mining led to the discovery of additional gene cluster with α-KGD and trimodular NRPS as partner, which generates diverse fumiquinazolines.

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ungal peptidyl alkaloids are a large group of natural products with diverse structure characterized by the presence of multicyclic, constrained architectures that can lead to high affinity for biological targets.1 The core of fungal peptidyl alkaloids is biosynthesized by nonribosomal peptide synthetases (NRPSs). Complexity and diversity generation in naturally occurring peptide alkaloids occur mainly through tailoring enzymes that catalyze redox reactions. A unique example of complexity generation from amino acid precursors can be found in the fumiquinazoline family of natural products, which contains a pyrazino[2,1-b]quinazoline-3,6-dione core linked to an indole moiety.2 Representative examples of fungal fumiquinazolines include alanditrypinone (1), fumiquinazolines F (FQF, 2), FQJ, FQA, FQC, and FQD with complex and diverse scaffolds (Figure 1). Structurally, FQF represents the least modified skeleton in this family. Other complex analogues are generated by coupling of varied additional amino acids and/or formation of strained bonds to afford multicyclic structures (Figures 1 and S1−S4). For example, coupling alanine to the indole side chain of FQF yields the imidazolindolone-containing FQA, and further connection between C3 and C17−OH or between C3 and N19 leads to spirohemiaminal FQC and cyclic aminal FQD, respectively. Alternatively, C−C bond connection through C3−C17 or C3− C18 lead to siproquinazolines and FQJ, respectively. The unique structures of fumiquinazolines result in promising bioactivities especially as chemotherapics, and the bioactivities © XXXX American Chemical Society

Figure 1. Representative fumiquinazolines with diverse skeletons. FQF represented the basic skeleton with reactive positions labeled in green.

are highly dependent on their stereochemistry at C3, C14, C17, and C18.2 Biosynthetically, a trimodular NRPS with three adenylation domains that incorporates anthranilate (Ant) and two varying amino acids as building blocks assembles the basic pyranoReceived: January 21, 2019

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DOI: 10.1021/acs.orglett.9b00260 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters

Figure 2. f mq-like gene clusters and their heterologous expression in A. nidulans. (a) aldp (i), ctq (ii), and f mq (iii) biosynthetic gene clusters in fungi. (b) LCMS analyses of culture extracts from heterologous coexpression of aldp and ctq genes. (c) Proposed biosynthetic pathway of fumiquinazolines.

quinazoline structure found in 2.3−6 More complex fumiquinazolines are constructed by additional enzymatic reactions that modify the FQF scaffold. The reactive positions at C3 and indolyl C17−C18 in the FQF molecule are specifically targeted for postmodification by redox enzymes. Two kinds of redox enzymes, FAD-dependent monooxygenases (FMO) and FADdependent pyrazinone oxidases, have been identified to generate diversity and complexity.1 For instance, a FMO (FmqB, Af12060) is involved in the indole epoxidation at C17−C18 of FQF, which is the site for incorporating an additional amino acid catalyzed by a monomodular NRPS (FmqC, Af12050) to form FQA. Alternatively and sometimes in parallel, a FAD-dependent oxidase (FmqD, Af12070) oxidizes the pyrazinone ring to a cyclic imine that undergoes intramolecular addition reactions by N and O nucleophiles3 (Figure S5). These modifications showcase how Nature uses redox enzymes to activate the FQF structure to generate multicyclic natural products. It is therefore of interest to identify novel strategies and enzymes Nature uses to synthesize other members of this family, especially the more complex structures observed in spiroquinazolines such as 1. Alanditrypinone (1) is the most structurally complex spiroquinazoline with unassigned absolute configuration and was initially isolated from an endophytic fungus Eupenicillium sp.7 Structurally, 1 possess a pyrazino-quinazolinedione tricyclic framework fused with a 6/5/5 aza-hybrid tricyclic fragment via C14−C15 and C3−C17 bonds to form the unique bridge-spiro coupled skeleton at sp3 chiral C17. Due to its unique complex structure, the total synthesis of 1 had not been reported. Here, we revealed the concise biosynthesis of 1

catalyzed by only three enzymes, a trimodular NRPS, a monomodular NRPS, and an α-ketoglutarate-dependent dioxygenase (α-KGD). We demonstrate the use of an αKGD enzyme is a new strategy to increase structural complexity starting from FQF. To investigate the biosynthetic pathway of 1, we sequenced the producer Penicillium janthinellum which was previously isolated from the leaf of Dracaena cambodiana (Figure S6). Bioinformatics analysis led to the identification of only one f mq-like NRPS gene cluster (named aldp cluster) in its genome (Figures S7−S9). The aldp cluster encodes an FmqAlike trimodular NRPS (AldpA) and a monomodular NRPS (AldpB) (Figure 2a, trace i). Compared to FmqA4 (Figure 2a, trace iii) and TqaA8 (Figure S5) which both contain an epimerase (E) domain in the second modular, AldpA does not contain an E domain in its domain sequence A-T-C-A-T-C-AT-CT. AldpA was proposed to activate anthranilate (Ant), and two other amino acids, to yield a tripeptide start with Ant (Figure S8 and Table S4). The absence of E domain in AldpA indicates the configuration of the second amino acid in the product of AldpA should be different from that in 2. In addition, an α-KGD (AldpC) instead of FAD-dependent oxidase exists in aldp gene cluster, containing the conserved potential HXDXnH motif that coordinates the iron cofactor (Figure S10).9 The presence of α-KGD as the only tailing redox enzyme in the aldp gene cluster suggests it may be multifunctional: (1) responsible for the C−C coupling to form the bridge-spiro coupled framework and (2) provide an oxidized intermediate for AldpB to load the second L-Trp moiety. B

DOI: 10.1021/acs.orglett.9b00260 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters To verify the aldp gene cluster and understand the function of each gene, different combinations of aldpA, aldpB, and aldpC were heterologously expressed in Aspergillus nidulans, and the resulting products were analyzed by LC−MS (Figure 2b, traces i−iv). Expression of aldpA alone in A. nidulans led to the production of 3 (Figure 2b, trace i, which possess same molecular weight to that of 2. Full structural characterization of 3 by 1D and 2D NMR (Figure S11 and Tables S6) confirmed it is 14-epi-FQF (i.e., ent-FQG),10 with the same planar structure to 2 but different in C14 configuration. The full structure of 3 with 14S configuration was consistent with the predicted domains in the trimodular NRPS, where the lack of E domain in AldpA would result in the stereochemical retention of L-Trp. Coexpression of aldpA and aldpB did not produce additional products besides 3 (Figure 2b, trace ii), which suggested that AldpB, the monomodular NRPS, cannot directly modify 3. We then coexpressed aldpA and aldpC in A. nidulans (Figure 2b, trace iii), which led to the generation of four new products 4−7. Compounds 4 and 5 were characterized as new compounds with hydroxyl group at C3, and 6 and 7 were identified as known compounds, dehydrofumiquinazoline and ent-FQJ, respectively.10 The final product alanditrypinone (1) along with minor products 3−7 were produced when aldpA, aldpB, and aldpC were heterologously coexpressed in A. nidulans (Figure 2b, trace iv). On the basis of the NMR data (Table S5) and the domain feature in AldpA, the absolute configuration of 3S,14S,18R,27S in 1 was reassigned as shown in Figure 2. This result confirms only three genes from the aldp gene cluster are required to complete the biosynthesis of a heptacyclical spiroquinazoline. To determine if any of 3−7 are biosynthetic intermediates to the formation of 1, we fed 3−7 separately to A. nidulans coexpressing aldpB and aldpC. The production of 1 was observed only when 3 was supplemented, which suggested 3 is an on-pathway intermediate to 1 and others are shunt products (Figures S12−S13). These results combined with those from heterologous expression suggest the generation of 1 from 3 requires AldpC to catalyze the formation of an iminium species such as 3a, which can be further modified by the additional LTrp moiety catalyzed by AldpB (Figure 3b).

weight and similar retention time to 4 in the LC−MS trace (Figures 3a and S15). Compound 4′ showed a very similar characteristic UV absorption to that of 1 but different from those of compounds 3−7 (Figure S16), indicating a spiroquinazoline scaffold for 4′. The polar nature and molecular weight of 4′ suggested it is most likely the hydrate of spiroquinazoline iminium containing intermediate 3a (Figure 3b). As expected, when the assay was performed in H218O buffers, we observed an increase in the molecular weight of 4′ by 2 mu. Attempts to purify this compound resulted in its rapid conversion to 4, 5, and 7 (Figure S17). In the absence of AldpB for loading the additional Trp, this unstable intermediate could convert to shunt products. Since 4 is shown to be an off-pathway shunt product in the biosynthesis of 1, generation of 3a via 4 by dehydration and successive arrangement is not reasonable. We proposed two possible mechanisms to form 3a from 3 catalyzed by AldpC (Figure 4): (i) abstraction of C3 hydrogen to give radical 3b,

Figure 4. Proposed catalyzed mechanism of AldpC. Red arrows represent the preferred mechanism.

further hydrogen abstraction at N19, and C−C radical coupling between C3 and C17 lead to the spiroquinazoline 3e, which can be protonated to form the 3a, and (ii) dehydrogenation of 3 to generate the ketoimine 3f and the protonated form of 3f (3g) undergo intramolecular attachment of C17 to C3 to generate 3a. Comparing the two pathways, the O atom at C3−OH in the shunt product 4 could derive from molecular oxygen in the case of pathway i, while derives from water in the case of pathway ii (Figures 4 and S13). After substrate radical formation, the OH group rebound generated the corresponding hemiaminal (4). It then eliminated the OH group to form the iminium intermediate. During incubation with H218O, attack of H218O will reform the hemiaminal (4 and 4a) and introduce the 18O as shown in Figure S15. When the same AldpC assay was conducted in complete H218O buffer with different reaction time, we observe a gradual increasing of incorporation of an O18 atom for 4 (Figure S15), suggested that the O atom at C3−OH in 4 was initially originated from 16 O2 and was changed to 18O in the H218O buffer. These results rule out the possibility of pathway ii, where C3−OH should all come from 18O in H218O buffer. As a result, the mechanism involved in radical oxidative cyclization (Figure 4a, path (i) is preferred for AldpC. As a result, AldpC was identified as a new example of nonheme iron α-KG-dependent oxygenase, which was involved in radical oxidative cyclization through C−C coupling in the biosynthesis of fungal natural products.11 We proposed that the shunt products 4 and 5 were generated from radical 3b by rebounding of the [OH•] from

Figure 3. In vitro assay of AldpC: (a) LCMS traces from in vitro assay; (b) proposed on-pathway intermediate 3a generated from 3.

To identify the missing on-pathway intermediate and reveal the enzymatic mechanism of AldpC, the soluble AldpC protein was expressed and purified to homogeneity from Escherichia coli BL21 (DE3) (Figure S14). Incubation of AldpC with 3 in the presence of α-KG and FeSO4 led to the generation of 4−7, along with an additional product 4′ with same molecular C

DOI: 10.1021/acs.orglett.9b00260 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters the resulting FeIII−OH, which led to insertion of the oxygen atom at C3. While shunt product 6 could be derived from 4 or 5 by dehydration and 7 derived from arrangement of 3a or 4 (Figure S17). To examine the catalytic promiscuity of AldpC, we examined if it also accepted FQF (2), a C14 epimer of 3, as substrate. No transformation was observed from the coincubation of AldpC and 2 in the presence of α-KG, Lascorbic acid, and FeSO4 (Figure 3a, trace viii). This result illustrated that AldpC is stereospecific to the substrate 3 and is consistent with its pairing with AldpA that lacks the E domain. Using AldpC as a lead, we examined the potential f mq-like cluster containing both α-KGD and trimodular NRPS such as AldpA in the genome database and found a target gene cluster (ctq) in the genome of Neosartorya f ischeri NRRL181 (Figure 1a, trace ii). The ctq cluster contains genes encode a trimodular NRPS (NFIA_057960, CtqA) without an E domain, a monomodular NRPS (NFIA_057990, CtqD), a FAD binding monooxygenase (NFIA_057970, CtqB) which is highly homologous to FmqB (Af12060, Figure S18), and an αKGD (NFIA_057980, CtqC) (Figure S6). Previously, Walsh et al. proposed that this gene cluster is responsible for the biosynthesis of fiscalins, which harbor the 14R configuration in the precursor.1 Based on the bioinformatics analysis and our previous results from AldpC assay, we predicted the ctq cluster is responsible for the biosynthesis of FQC/FQD-like compounds with 14S other than 14R configuration. Indeed, expression of ctqA alone led to the production of 8 (Figure 2b, trace v), which was identified as (+)-glyantrypine. Glyantrypine was initially isolated from Aspergillus clavatus with unknown absolute configuration.12 Coexpression of ctqA/B/D formed 9 (Figure 2b, trace vi), which was characterized as known compound fumiquinazoline Q.13 Moreover, coexpression of ctqA/B/C/D generated known compound cottoquinazolie E14 (10) and a new compound 11, named cottoquinazolie G (Figure 2b, trace vii). Full structural characterization of 8−11 was carried out by detail 1D and 2D NMR analysis. Comparing to fumiquinazolines F, A, C, and D, compounds 8−11 miss a methyl group at C3 and do possess the 14S rather than the 14R configuration. The identification of ctq cluster (Figure S6) and its corresponding products further supported the partner of α-KGD and trimodular NRPS without E domain in the biosynthesis of fumiquinzoline alkaloids. Therefore, our discovery (1) reveals the biosynthetic diversity of this family of fungal peptidyl alkaloids where an a-KGD enzyme is used as new strategy to increase structural complexity; (2) provide a way to solve the unassigned and confused stereochemistry of fumiquinzolines based on the presence or absence of E domain in trimodular NRPS; and (3) accelerate the genome mining guided discovery of diverse fumiquinzolines. In summary, the complexity and diversity of fumiquinazoline related alkaloids were mainly generated from building blocks of anthranilate (Ant) and varying amino acids, as well as additional redox modification. The presence or absence of E domain in the trimodular NRPS increase the diversity of stereochemistry. The trimodular NRPS assembling FQF contributes for the first level of complexity. In the case of presence of FMO (such as FmqB, TqaH, and CtqB) in the gene cluster, FMO will function first at the reactive indolyl C17−C18 position of the FQF scaffold to form an epoxide, which is ready to couple an additional amino acid to yield the imidazolindolone containing FQA-like compound. Finally the FAD-dependent pyrazinone oxidase (pairing to E domain

containing NRPS) or a-KGD (pairing to E domain free NRPS) introduces a third layer of scaffold complexity by converting FQA to the spirohemiaminal FQC and FQD-like compounds, presumably by catalyzing the formation of a transient imine within the pyrazinone ring. While in the case of no FMO existing in the gene cluster, an α-KGD pairing to E domainfree trimodular NRPS will function before the monomodular NRPS at the reactive C3 position to generate an iminiumcontaining intermediate, which was finally convert to spiroquinazolines with a 14S configuration by loading the latter amino acid (Figure S68). We propose the alanditrypinone-like compounds with the 14R configuration, such as spiroquinazoline, are biosynthesized by a gene cluster containing three genes encoding a trimodular NRPS with E domain, a monomodular NRPS, and a FAD-dependent pyrazinone oxidase (or other type of redox enzyme), which exists in Nature and has yet to be uncovered. In conclusion, we elucidated the concise three enzyme biosynthetic pathway of heptacyclical alanditrypinone, where an α-KGD catalyzed the radical oxidative cyclization through C−C coupling and provided an iminium containing intermediate for loading an additional amino acid. Moreover, we further demonstrated that the α-KGD and trimodular NRPS without E domain acted as partner leading to the diversity generation in the fumiquinazolines biosynthesis. Our discovery opens a new window for mining and combinational biosynthesis of fumiquinazoline and structurally related peptidyl alkaloids

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b00260. Full experimental details and spectroscopic data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Youcai Hu: 0000-0002-3752-7485 Author Contributions §

D.Y., Q.C., and J.G. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported financially by the CAMS Innovation Fund for Medical Sciences (CIFMS, 2017-I2M-4-004), the Drug Innovation Major Project (2018ZX09711001-006), and Fundamental Research Funds for the Central Universities (2017PT35001); Q.C. was supported by the Postdoctoral Science Foundation of China (2018M631399). We are grateful to Prof. Wenbin Yin (Institute of Microbiology, Chinese Academy of Sciences, Beijing, China) for sharing Aspergillus nidulans.

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REFERENCES

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DOI: 10.1021/acs.orglett.9b00260 Org. Lett. XXXX, XXX, XXX−XXX