Identification of the Amipurimycin Gene Cluster Yields Insight into the

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Identification of the Amipurimycin Gene Cluster Yields Insight into the Biosynthesis of C9 Sugar Nucleoside Antibiotics Wen-Jia Kang,†,§ Hai-Xue Pan,‡,§ Shengyang Wang,‡ Biao Yu,‡ Huiming Hua,*,† and Gong-Li Tang*,‡ †

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Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China ‡ State Key Laboratory of Bio-organic and Natural Products Chemistry, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences (CAS), CAS, Shanghai 200032, China S Supporting Information *

ABSTRACT: Feeding studies indicate a possible synthetic pattern for the N-terminal cis-aminocyclopentane carboxylic acid (ACPC) and suggest an unusual source of the highcarbon sugar skeleton of amipurimycin (APM). The biosynthetic gene cluster of APM was identified and confirmed by in vivo experiments. A C9 core intermediate was discovered from null mutants of ACPC pathway, and an ATP-grasp enzyme (ApmA8) was reconstituted in vitro for ACPC loading. Our observations allow a first proposal of the APM biosynthetic pathway.

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mipurimycin (APM, 1) is a complex peptidyl nucleoside antibiotic originally isolated from Streptomyces novoguineensis sp. nov. in 1977.1,2 It shows remarkable activity against Pyricularia oryzae, which is known to cause the rice blast disease.1 On the basis of chemical degradation and extensive spectroscopic analysis in 1982,3 the primary structure of APM was reported, which consists of (a) a unique C9 pyranosyl amino acid bearing a two-carbon branch on C-3′, (b) a glycosidic β-linked 2-amino-purine, and (c) an N-terminal cisaminocyclopentane carboxylic acid (ACPC, 3) residue (Figure 1A). However, the stereochemistry at C-6′ and the absolute configuration at C-2′′/C-3′′ have not been determined.3 The structural features, as well as the uncharacterized mechanism of antifungal action, render APM an attractive yet challenging target for synthesis. In the past three decades, various structural fragments and analogues have been synthesized.4−12 However, the true configuration of APM was not resolved until a recent work on total synthesis of eight diastereoisomers, where the authors suggested that the configuration previously assigned at C-3′ should be opposite.13 The challenges in total synthesis of APM have also raised interest in its biosynthesis. However, both the precursors and the biosynthetic pathway of APM remain unresolved. Based on the retrosynthetic analysis, the branched C9 skeleton of APM is most likely constructed by an aldol condensation between a pentose and a threonine (Thr) derivative (Figure S1), despite the lack of similar enzymatic reactions.13,14 In addition, pentose could react with Thr to form a C7 sugar amino acid (C-1′−C-7′) and an acetaldehyde, similar to the reactions catalyzed by serine hydroxymethyltransferase (SHMT)-like proteins (Figure S2).15−18 Likewise, © XXXX American Chemical Society

Figure 1. (A) Originally proposed and revised structure of APM. (B) Organization of the apm biosynthetic gene cluster.

the C9 or C7 skeleton could be obtained through carbon extension by a radical S-adenosyl-L-methionine (SAM) enzyme19−22 or polyketide synthase (PKS). To obtain clues about the possible biosynthetic pathways for 1, we first performed a feeding experiment with [1,2-13C]acetate followed by APM isolation and 13C NMR analysis. Surprisingly, no significant 13C enrichment of the C9 skeleton was observed (Figure 2A), indicating that this high-carbon sugar amino acid should not be constructed between pentoses Received: March 7, 2019

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DOI: 10.1021/acs.orglett.9b00840 Org. Lett. XXXX, XXX, XXX−XXX

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an intact C-2−C-3 unit of pyruvate.25,26 An additional feeding experiment for APM with U-13C-pyruvate showed the same result as [1,2-13C]-acetate (Figure S4). Therefore, we hypothesized that the biosynthesis of ACPC in APM adopts a PKS pathway similar to the CPC path, but their C4 precursor may be derived from different blocks. A feeding experiment with [U-13C]-ribose showed that the C9 core skeleton is not derived from an intact ribose (Figure S5). To the best of our knowledge, this is rare in the biosynthesis of high-carbon sugar nucleosides. Additional experiments with [U-13C]-glucose and [1,2-13C2]-glucose indicated that C-1′−C-3′ and C-4′−C-6′ of APM are derived from two different metabolites, respectively (Figures S6−S8). Due to the conversion from ribose to glucose, the feeding results with two precursors are consistent with each other (Figure S8). Combined with the feeding results of 13C-labeled acetate, it is proposed that C-1′−C-3′, C-4′−C-6′, C-8′−C-9′ and C-7′ of APM are derived from several metabolites of Dglucose, respectively, which do not include those produced in or after the citric acid cycle. The genome of S. novoguineensis sp. nov. was sequenced, and then the antiSMASH server and local BLAST approach were utilized to search for possible targets based on the feeding results.27 One locus with clustered genes (apmA1−A7) was discovered when Cfa3, a β-ketoacyl-ACP synthase (KS) in the CPC pathway, was used as an inquiry (Figure 1B).24,28,29 The locus includes the following: apmA1 encoding a long-chain fatty acid-CoA ligase (43% identity to Cfa5); apmA2, a hypothetical protein (51% identity to Cfa4); apmA3, a KS (53% identity to Cfa3); apmA4, a β-hydroxyacyl-ACP dehydratase (62% identity to Cfa2); apmA7, an acyl carrier protein (ACP, 46% identity to Cfa1); apmA5 and apmA6, encoding an acyl-CoA thioesterase and an aminotransferase, respectively (Figure 4A and Table S1). The deduced enzymes highly meet the requirements of ACPC synthesis. Subsequent analysis of the genomic context surrounding apmA1−A7 revealed 29 clustered genes ranging from apmA1 to apmR3, spanning 32.5 kb of DNA, which were hypothesized to harbor the structural genes for APM biosynthesis (named apm, Figure 1B and Table S1). During our study, the limited yield of APM in fermentation hinders the efficiency of in vivo experiments (Figure 3A, lane i). In the apm cluster we noticed two positive regulatory genes, apmR1 and apmR2, which encode SARP family transcriptional regulators that usually function as pathway specific activators. In order to improve APM titer, apmR1 and apmR2 were cloned into pSET152 under the control of an ermEp* promoter and then introduced into S. novoguineensis (WT) to obtain the chromosomally integrated recombinant strains, APMR1 and APMR2, respectively. Compared with WT, the production of APM by APMR1 and APMR2 was improved; in particular, the yield in the APMR2 strain was significantly increased by 30-fold (Figure 3A, lanes ii−iii). Therefore, in the subsequent experiments, we introduced the ermEp*-apmR2 cassette into the genome of each gene knockout mutant to facilitate the discovery of intermediates. To confirm that the apm cluster is involved in APM biosynthesis, an apmB6-deletion mutant (ΔapmB6::apmR2) was selectively constructed by in-frame deletion. ApmB6 is a nonribosomal peptide synthetase (NPRS)-like protein that contains an adenylation domain (A) and a peptidyl carrier protein (PCP), presumably playing a key role in the synthesis of the C9 skeleton. By LC-MS analysis, the apmB6-deletion

Figure 2. 13C NMR spectra of APM isolated from S. novoguineensis sp. nov. fed with 13C-labeled sodium acetate. (A) The culture was fed with (I) no additive, (II) [1,2-13C]-acetate, and (III) [1-13C]-acetate. (B) The multiplicity analysis of C-1′′ to C-4′′ in APM fed with [1,2-13C]-acetate.

and Thr derivatives because acetate can be incorporated into Thr in primary metabolism (Figures S1 and S2). This result also excludes the possibility that the C9 core is formed by PKS employing Claisen condensation with malonyl-CoA (M-CoA) as an extender unit. On the other hand, remarkable enrichment was observed at all of the carbons of the ACPC moiety, four of which are derived from intact acetate (C-1′′−C-2′′ and C-3′′−C-4′′) (Figure 2). Subsequent experiments showed that the 13C enrichment of APM fed with [1-13C]-acetate occurred only at C-1′′ and C-3′′ (Figure 2). It means that the C6 backbone of ACPC is derived from neither a common polyketide nor a fatty acid containing three intact acetates.23 Therefore, a C4 compound was presumably the source of C-3′′ to C-6′′ of ACPC, which is most likely derived from the α-ketoglutarate formed in the tricarboxylic acid cycle (Figure S3). It is noted that an intermediate of coronafacic acid (CFA), 2-carboxy-2cyclopentenone (CPC), was proposed to be synthesized by a PKS from the α-ketoglutarate precursor.24 However, our feeding result is inconsistent with that for CFA because C-5 and C-6 of CPC in CFA are not derived from acetate but from B

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in the ApmB5-catalyzed extension, which is responsible for activating and carrying a carboxylic acid or amino acid, while the potential substrate of the A domain of ApmB6 cannot be predicted from its substrate specificity-conferring codes (Table S1 and Figure 4C).31,32 In combination with the feeding results, the C7 linear core (C-1′−C-7′) of APM is proposed to be formed by a two-carbon extension on the ApmB6 activated C6 substrate catalyzed by ApmB5, followed by decarboxylation. The presence of ApmB1−B4 suggests that the extender unit utilized by ApmB5 may be HM-ACP instead of M-CoA, which is consistent with the feeding results (Figure 4B and 4C). Furthermore, we found two-component transketolases (TKases), ApmC1 and ApmC2. ApmC1 shows high homology with the thiamin pyrophosphate (TPP) binding domain, and ApmC2 contains a pyrimidine (PYR) binding domain and a Cterminal domain of TKase. In general, TKases are responsible for the transfer of the C2 fragment from ketose to aldose or to a downstream enzyme.33,34 Therefore, the subsequent introduction of C-8′−C-9′ is presumed to be catalyzed by the TKases ApmC1/C2 from a glucose derivative (Figure 4C). There are no other enzymes encoded by the apm gene cluster that might catalyze the formation of C−C bonds. The cluster also encodes enzymes with well-defined functions or enzymes that should play important roles in APM biosynthesis, but their functions cannot be assigned by bioinformatic analysis (Table S1). A branched-chain amino acid aminotransferase, ApmJ, is probably responsible for the formation of an amino group at C-6′. A kinase, ApmH, presumably catalyzes the phosphorylation of the C9 intermediates at C-1′, which may be necessary for the linkage between the C9 unit and purine. There are two SDR family oxidoreductases, ApmE and ApmM, a FAD-dependent oxidoreductase, ApmG, and a Gfo/ Idh/MocA family oxidoreductase, ApmK, which are presumed to be responsible for the redox process of the C9 backbone, 1ene-ACPC and guanine. ApmA8 is an ATP-grasp domaincontaining protein which is usually responsible for the generation of an amide bond in some cases,35 presumably catalyzing the linkage between ACPC and the C9 sugar amino acid (Figure 4C). However, there are a few reactions in the APM biosynthetic pathway that cannot be attributed to specific enzymes, such as hydrolysis of the C8 intermediate and subsequent decarboxylation, and the attachment of 2-aminopurine to the C9 backbone. Candidate enzymes for these processes may include a methyltransferase, ApmF; a TIGR00730 family Rossman fold protein, ApmD; and two hypothetical proteins, ApmI and ApmL. It was speculated that apmA1−A7 and an adjacent gene apmA8 are responsible for the formation and attachment of ACPC. To verify this hypothesis, these genes were selectively knocked out. As predicted, the production of 1 was completely eliminated in the resulting strains except for apmA5 mutant (ΔapmA5::apmR2), while an unidentified metabolite (2) accumulated in a large amount, which shows an identical UV spectrum to APM (Figure 3A, lanes v−ix, Figure S9). Structural characterization of compound 2 isolated from the ΔapmA3::apmR2 mutant by HRMS and NMR analysis indicated that it is identical to the nucleoside portion of APM without ACPC residue (named APM-384) (Figure 3C, Table S2 and Figures S10−S18). A small amount of APM was still produced in the apmA5 deletion mutant, probably because the hydrolytic function of ApmA5 may be taken over by other

Figure 3. (A) LC-MS profiles of WT (i), APMR1 (ii), APMR2 (iii), ΔapmB6::apmR2 (iv), ΔapmA1::apmR2 (v), ΔapmA3::apmR2 (vi), ΔapmA4::apmR2 (vii), ΔapmA5::apmR2 (viii), and ΔapmA8::apmR2 (ix) cultures. (B) LC-MS profiles of in vitro assays for ApmA8: authentic standards (i); ApmA8-catalyzed reaction with 2 and 3 as substrates in the presence of ATP and MgCl2 (ii); reactions without ApmA8 (iii), ATP (iv), and MgCl2 (v). (Extracted ion chromatograms in A and B: 496.2 for 1, 385.2 for 2, 130.0 for 3.) (C) Key 2D NMR correlations of 2.

mutant completely lost the ability to produce APM (Figure 3A, lane iv). This result determined the correlation between the apm cluster and APM biosynthesis. Functional assignment for apm genes showed that the putative pathway is quite different from other nucleoside biosynthetic pathways. ApmB1−B4 share homology with four enzymes involved in the biosynthesis of (2R)-hydroxymalonylACP (HM-ACP) from 1,3-bisphosphoglycerate (1,3-BPG) and are likely responsible for the synthesis of this PKS extender unit (Table S1). The proposed pathway for HM-ACP is as follows (Figure 4B): (i) ApmB1, a FkbH like protein, dephosphorylates 1,3-BPG and tethers it to ApmB3 forming a glyceryl-ACP; (ii) ApmB4, a 3-hydroxyacyl-CoA dehydrogenase, catalyzes the oxidation of glyceryl-ACP to 2-hydroxy-3oxopropionyl-ACP; and (iii) ApmB2, an acyl-CoA dehydrogenase, converts the ApmB4-product to HM-ACP (6).30 ApmB5 is a three-domain PKS containing KS, acyltransferase (AT), and ACP, in which the catalytic triad (C−H−H) of KS indicates an intact condensation function for a two-carbon extension. The NRPS-like ApmB6 is the most likely participant C

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Figure 4. Summary of APM biosynthesis. (A) Proposed ACPC pathway. (B) Proposed pathway for (2R)-hydroxymalonyl-ACP formation. (C) A putative biosynthetic route for the branched C9 sugar and the final assembly of APM.

In summary, feeding results using 13C-labeled precursors indicate that the biosynthetic pathway of the C9 branched sugar in APM is different from that of other high-carbon nucleosides. The biosynthetic gene cluster was identified from the genome based on the determination of the precursors for ACPC biosynthesis and subsequently verified by gene deletion experiments. By combining the genetic composition of the cluster and the feeding results, a putative PKS-dependent pathway for C9 core formation was proposed. A new APM intermediate, APM-384, was discovered by genetic manipulation of regulators and inactivation of the ACPC related genes. In addition, the catalytic activity of ApmA8 was reconstituted in vitro using APM-384 and chemically synthesized ACPC as substrates. This study sets the stage for further deciphering of the enzymatic mechanism for assembly of this class of highcarbon nucleoside natural products.

hydrolases encoded outside the apm cluster or the hydrolysis proceeds spontaneously. To further explore the installing of ACPC to the C9 core, the protein ApmA8 was overexpressed in Escherichia coli BL21(DE3) and purified as a N-His6-tagged protein (Figure S19), and ACPC (3) was chemically prepared as the amino acid donor. Enzymatic assays indicated that ApmA8 could catalyze the loading of 3 onto 2 to form 1 (Figure 3B, lanes ii− iii). The reaction requires the participation of ATP and Mg2+, and ADP is produced after the reaction (Figure 3B, lanes iv−v and Figure S20), which is consistent with the mechanism through an acyl-phosphate intermediate for ATP-grasp enzymes.35 The result suggests that the attachment of the ACPC unit to 2 should be the final step in the biosynthesis of APM. D

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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.9b00840. Material and methods, supplementary tables and figures (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Biao Yu: 0000-0002-3607-578X Huiming Hua: 0000-0002-0258-3647 Gong-Li Tang: 0000-0003-3149-4683 Author Contributions §

W.-J.K. and H.-X.P. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (81473124 and 21621002), the Science and Technology Commission of Shanghai Municipality (18ZR1448500), and the CAS (Youth Innovation Promotion Association 2016235, XDB20000000, QYZDJSSW-SLH037 and K. C. Wong Education Foundation).

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