Sparsomycin Biosynthesis Highlights Unusual Module Architecture

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Sparsomycin Biosynthesis Highlights Unusual Module Architecture and Processing Mechanism in Non-ribosomal Peptide Synthetase Zhe Rui, Wei Huang, Fei Xu, Mo Han, Xinyu Liu, Shuangjun Lin, and Wenjun Zhang ACS Chem. Biol., Just Accepted Manuscript • Publication Date (Web): 05 Jun 2015 Downloaded from http://pubs.acs.org on June 8, 2015

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ACS Chemical Biology

Sparsomycin Biosynthesis Highlights Unusual Module Architecture and Processing Mechanism in Non-ribosomal Peptide Synthetase Zhe Rui,†,§ Wei Huang,†,§ Fei Xu,‡,§ Mo Han,‡ Xinyu Liu,¶ Shuangjun Lin,*,‡ and Wenjun Zhang*,†,⊥ ‡

State Key Laboratory of Microbial Metabolism, Joint International Laboratory on Metabolic & Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China †

Department of Chemical and Biomolecular Engineering, 201 Gilman Hall, MC 1462, University of California, Berkeley, CA 94720, USA ¶

Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, PA 15260, USA

⊥Physical

Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

Supporting Information Placeholder

ABSTRACT: Sparsomycin is a model protein synthesis inhibitor that blocks the peptide bond formation by binding to the large subunit of ribosome. It is a unique dipeptidyl alcohol, consisting of a uracil acrylic acid moiety and a monooxo-dithioacetal group. To elucidate the biosynthetic logic of sparsomycin, a biosynthetic gene cluster for sparsomycin was identified from the producer Streptomyces sparsogenes by genome mining, targeted gene mutations, and heterologous expression. Both the genetic and enzymatic studies revealed a minimum set of non-ribosomal peptide synthetases needed for generating the dipeptidyl alcohol scaffold of sparsomycin, featuring unusual mechanisms in dipeptidyl assembly and offloading.

Sparsomycin, a secondary metabolite from Streptomyces sparsogenes, displays a rare broad-spectrum antibiotic and antitumor activity against bacteria, archaea, eucarya, and 1 various cancer cell lines. Sparsomycin inhibits the peptide bond formation in the protein synthesis by specifically binding to the A-site of the large subunit of ribosome and stabilizing the binding of tRNAs to the P-site, for which reason it has also been widely used as a powerful tool to study the 2-3 protein synthesis. The molecular structure of sparsomycin features two unusual subunits, 6-methyl-uracil acryloyl moiety and the oxo-methylthiomethyl-cysteinol (O-MTMcysteinol) moiety that are bridged by an amide bond (Figure 1). These unique chemical functionalities render sparsomycin and its analogues important roles in the natural productbased small molecule library for drug discovery. The biosynthesis of sparsomycin has been a problem of particular interest due to its important biological activity and unusual structural features. Through labeled precursor feeding experiments, Parry and co-workers showed that the MTM-cysteinol moiety arises from L-Cys via the intermediacy of MTM-Cys, and the 6-methyl-uracil acryloyl subunit is de4 rived from L-Trp via 6-methyl-uracil acrylic acid (UAA). It

was further deduced that reduction of the carboxyl group of MTM-Cys to form MTM-cysteinol precedes the attachment of UAA. Later, the same group isolated the enzyme that catalyzes the conversion of 6-methyl-pyrimidine acrylic acid to UAA, although no sequencing information was reported; interestingly, this enzyme also displays weak xanthine dehydrogenase (XDH) activity in converting hypoxanthine to 5-6 xanthine. Despite decades of biosynthetic investigations, the genetic basis for sparsomycin biosynthesis remained unknown, which prevented the study of enzymatic reactions in transforming precursors such as L-Cys and L-Trp into the interesting compound sparsomycin. Toward this end, we here have undertaken the identification of the sparsomycin biosynthetic gene cluster from the producer organism S. sparsogenes through genome mining, targeted gene disruption, and heterologous expression. In addition, we have reconstituted the activity of the newly identified highly unusual non-ribosomal peptide synthetase (NRPS) machinery in generating the dipeptidyl alcohol skeleton of sparsomycin through in vitro enzymatic studies. For mining the sparsomycin biosynthetic genes, the genome of S. sparsogenes ATCC 25498 was subjected to Illumina sequencing which resulted in ~9.7 M non-redundant bases after assembly of paired sequence reads. A local BLASTP analysis was then performed using two enzyme probes based on the assumption that secondary metabolite biosynthetic genes are clustered in microbial organisms. These two probes include XDH and a possible amide-forming enzyme such as NRPS, amide synthetase, or ATP-grasp ligase. The in silico analysis yielded only one putative biosynthetic gene cluster in which genes encoding both XDH and NRPS are in close proximity. Further bioinformatics analysis revealed that this gene cluster spans ~30 kb and consists of 24 open reading frames (ORFs), here designated as spsA-X (Figure 1, Table S1). Of these ORFs, spsQ and spsR encode NRPSs including a total of three adenylation (A) domains, three thiolation (T) domains, one condensation (C) domain, and one epimerization domain (E). SpsD/E/F were assigned to convert 6-

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methyl-pyrimidine acrylic acid to UAA based on their homology to XdhA/B/C and CdhA/B/C, the heterotrimeric

UAA synthesis

other oxidoreductase

transporter

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plasmid through the DNA assembler system in yeast (Figure 1 11 and Figure S3). The resulting plasmid, pZR94, was se-

NRPS related

MTM-Cys synthesis

Unknown

Figure 1. Map of the sparsomycin gene cluster and proposed biosynthetic pathway. xanthine dehydrogenase and caffeine dehydrogenase that catalyze the oxidation of hypoxanthine to xanthine in E. coli and caffeine to 1,3,7-trimethyluric acid in Pseudomonas, re7-9 spectively. Concomitantly, we pursued the sparsomycin biosynthetic gene cluster discovery through heterologous expression of the cosmid library of S. sparsogenes in the sparsomycinsensitive strain S. lividans TK24, followed by sparsomycinresistant clone selection (Figure S1). The underlying rationale for this method is that biosynthetic genes and genes conveying resistance for a natural product are typically co-localized in the microbial genome. The sequencing of the resulting cosmid led to the identification of the same putative sps cluster region as predicted through genome mining. SpsK and spsL, the genes encoding homologs of a multidrug resistance efflux pump and an ABC transporter respectively, likely convey the sparsomycin resistance in S. lividans. To confirm the role of the sps cluster in the biosynthesis of sparsomycin, we first attempted to create gene disrupted mutants of S. sparsogenes followed by metabolic profiling. The knockout targets include spsQ, encoding an A-T didomain NRPS, and spsR, encoding an A1-T1-C-A2-T2-E hexadomain NRPS. Both genes were deleted in-frame through double crossover according to standard Streptomy10 ces genetics protocol, and the resulting mutants were confirmed by PCR (Figure S2). The production of sparsomycin was abolished in both mutants, demonstrating that these NRPS genes are essential for the biosynthesis of sparsomycin (Figure 2a). In addition, we attempted to heterologously express the sps cluster in S. lividans K4-114 for a parallel in vivo confirmation of the cluster. Because we did not isolate a cosmid that contained the full length of the putative sps cluster, we assembled two overlapping cosmids (BE6 and AH1) with an E. coli-Streptomyces shuttle vector into a single

quenced to confirm that it contained the entire predicted sps biosynthetic gene cluster. pZR94 was then introduced into S. lividans by conjugation, and the culture of transconjugant was subjected to metabolite analysis. Surprisingly, no sparsomycin production by S. lividans-pZR94 was detected even with the very sensitive analysis of liquid chromatography– high resolution mass spectrometry (LC-HRMS) (Figure 2b). We then fed possible biosynthetic intermediates, such as UAA or MTM-Cys, to probe their ability in promoting the heterologous production of sparsomycin. While the feeding of UAA had no obvious effect, the addition of MTM-Cys to the fermentation culture enabled sparsomycin production in S. lividans-pZR94 (Figure 2b), suggesting the inadequacy of this sps cluster towards the formation of the MTM-Cys and the sufficiency of this cluster on the formation of UAA as well as the final conversion to sparsomycin in S. lividans. We therefore unambiguously verified that the identified sps cluster from S. sparsogenes is directly involved in the biosynthesis of sparsomycin through both the gene disruption and the heterologous expression experiments. We next scrutinized the NRPS machinery encoded by the sparsomycin gene cluster by in vitro enzymatic studies. This NRPS system is of particular interest for two reasons. First, sparsomycin is a dipeptide presumably assembled from two monomers, but a total of three A domains are encoded by spsQ and spsR, possibly capable of activating three monomers. Second, the mechanism for dipeptidyl alcohol formation remains elusive. It has been known that a reduction (R) domain of NRPS can utilize NAD(P)H to reductively release a polypeptide chain from NRPS to yield an aldehyde or 12-15 but no such R domain was identified from the alcohol, modular NRPSs encoded by spsQ and spsR. Furthermore, previous feeding results suggested that reduction of the carboxyl group of MTM-Cys precedes


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ACS Chemical Biology a.

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SpsQ

SpsR

100%

100%

80%

80%

60%

60%

40%

40%

20%

20%

0%

0%

2 1 b. HO

O

I S

II

Figure 2. Analysis of sparsomycin production from mutants of S. sparsogenes and heterologous host S. lividans K4-114. a) HPLC chromatograms with UV detection (302 nm) of metabolites produced by wild-type and mutant S. sparsogenes strains. I) Wild-type S. sparsogenes; II) ΔspsQ; III) ΔspsR; IV) ΔspsM; (V) ΔspsS. b) Heterologous production of sparsomycin. Extracted ion chromatograms showing I) production of sparsomycin by wild-type S. sparsogenes, II) and III) heterologous production of sparsomycin in S. lividans-pZR94 with or w/o supplementation of MTM-Cys, respectively. The calculated mass for sparsomycin with 10 ppm mass error tolerance was used. attachment of the uracil moiety, which is inconsistent with the NRPS-dependent mechanism for dipeptidyl alcohol formation. To gain insight into functions of the encoded NRPS machinery, we first assayed the ability of three A domains to reversibly adenylate various acids using the classical ATP32 16 [ P]PPi exchange assay. The intact proteins, SpsQ and SpsR, as well as the truncated SpsR, SpsR-A1 and SpsR-A2, were overexpressed and purified from E. coli BAP1 (Figure 17 S4). Since bioinformatics analysis failed to predict the substrates for these A domains, a broad range of substrates was tested (Figure 3a and S5). SpsQ exhibited a strong preference for activation of MTM-Cys over all other tested acids; SpsR and SpsR-A1 were demonstrated to specifically activate UAA; no substantial activation of any acids was detected in SpsRA2 assays, consistent with the observation that SpsR failed to activate MTM-Cys. In addition, the activation of MTM-L-Cys is in agreement with the presence of E domain, which presumably converts L- to D-configuration of this asymmetric center found in sparsomycin. The substrate preference of these A domains are also in a good alignment with the identity of two components of sparsomycin, but the function of SpsR-A2 domain remains unclear. Bearing one C domain based on sequence analysis, SpsR was postulated to catalyze the formation of amide linkage between the T domains tethered MTM-Cys and UAA residues. Indeed, in vitro assay with ATP, MTM-Cys, UAA, SpsQ, and SpsR yielded a trace amount of compound 1, which was revealed to be a dipeptidyl acid by LC-HRMS and HRMS/MS analysis (Figure 3b and S6). 1 is presumably formed through a spontaneous slow hydrolytic offloading reaction from NRPS. Control experiments showed that the production of 1 was dependent on both enzymes (SpsQ/R) and all three substrates. The requirement of SpsQ for 1 production is con32 sistent with the results of ATP-[ P]PPi exchange assays, and further demonstrated that the dimodule NRPS SpsR alone is insufficient for dipeptidyl assembly. To screen for enzymes reducing the dipeptidyl

S

1

III

O

O

N H

NH N H

O

IV V 7

9

11

13

15 17 t/min

Figure 3. Biochemical characterization of SpsQ/R/M. a) Adomain activities of SpsQ and SpsR. A relative activity of 100% for MTM-Cys and UAA-dependent exchange corresponds to 68k and 53k cpm, respectively. Refer to Figure S5 for a broader range of tested substrates. b) Extracted ion chromatograms showing the product formation in the reactions containing ATP, UAA, and MTM-Cys under the catalysis of I) SpsQ/R, II) SpsQ/R/M with NADH, III) SpsQ/R/M with NADPH, IV) SpsQ/M and SpsR(K1552A) with NADPH, and V) SpsQ/M and SpsR-A2t with NADPH. The calculated masses for 1 and 2 with 10 ppm mass error tolerance were used for each trace. intermediate to an alcohol, we then surveyed the activities of four putative reductases, SpsB, SpsJ, SpsM, and SpsU, encoded by the sparsomycin gene cluster (Table S1). The addition of SpsM, but not any other reductases, to the SpsQ/R reaction system yielded a new compound 2, which was shown to be a dipeptidyl alcohol that had previously been reported 18 (Figure 3b and S7). No dipeptidyl aldehyde was detected by extracted mass analysis. In addition, disruption of spsM in S. sparsogenes completely abolished the production of sparsomycin (Figure 2a), confirming the critical function of the encoding enzyme. Based on the sequence homology analysis, SpsM belongs to the family of short-chain dehydrogenas19 es/reductases (SDRs) which typically contains a conserved Y157XXXK161 active site motif and an NADPH binding site. As expected, omitting NADPH or substituting NADPH with NADH in the in vitro assays abolished the production of 2 (Figure 3b). Interestingly, the phylogenetic tree analysis of SpsM showed that this enzyme is evolutionarily far from the characterized R domains of NRPSs that catalyze the reductive release of thioester-bound peptidyl substrates, but rather close to the characterized SDRs that mostly perform 2e reduction on free-standing substrates (Figure S8). To exclude the possibility of 1 to be the direct substrate for SpsM, the enzymatic reaction for 1 formation was scaled-up and 1 was purified by HPLC. Incubation of 1, NADPH, and SpsM (with or without SpsQ/R/ATP) did not yield 2 (Figure S9), indicating that SpsM functions on the tethered dipeptidyl intermediate of the NRPS assembly line. We thus confirmed that SpsM is a cryptic stand-alone reductase capable of offloading a tethered peptidyl intermediate through [2+2]e reduction. Notably, the formation of hydroxyl group of sparsomycin occurs after the dipeptide assembly, contradicting to the



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previous claim that reduction of the carboxyl group of MTM4 Cys precedes attachment of UAA. 2 is proposed to be the direct precursor for sparsomycin formation prior to the final oxidation event that is likely catalyzed by SpsP, a cysteine dioxygenase homolog. The presence of three T domains encoded by spsQ and spsR suggests that aminoacyl group transfer is likely in the biosynthesis of dipeptide sparsomycin. Indeed, transfer of aminoacyl groups between T domains to facilitate subsequent tailoring has previously been observed in NRPS ma20-21 chinery. We propose that upon loading of MTM-Cys onto SpsQ, this moiety is transferred to SpsR-T2 for subsequent condensation and epimerization reactions (Figure 1). The transthiolation reaction could be spontaneous, such as in pacidamycin biosynthesis, or promoted by an acyltransferase of the α,β-hydrolase family, such as CmaE in coronamic acid 20-21 Only one acyltransferase candidate, SpsS, biosynthesis. was found encoded in the sps cluster, and the spsS disrupted mutant of S. sparsogenes failed to accumulate detectable amount of sparsomycin (Figure 2a). We then purified and introduced SpsS to the SpsQ/R/M in vitro assay, which resulted in >100-fold increase of 2 production (Figure S10), strongly indicating the role of SpsS in facilitating the transthiolation reaction between T domains. The reconstitution of enzymatic activities of SpsQ/R/M/S in generating the dipeptidyl alcohol skeleton of sparsomycin allowed us to further probe the necessity of A2 domain of SpsR in the NRPS machinery. Although SpsR-A2 failed to activate any tested acids, sequence alignment showed that a strictly conserved Lys (K1552) is present in this A2 domain, which is a universal feature of carboxylic acid activating ade22 nylation enzymes. We thus constructed an expression construct for SpsR bearing a point mutation of K1552A. Additionally, the whole predicted A2 domain was also removed to make a truncated SpsR-A2t. Both SpsR variants were expressed and purified with a comparable yield of soluble proteins as the wild type (Figure S4). Replacement of SpsR by these SpsR variants in the enzymatic assays of SpsQ/R/M/S completely abolished the production of 2 (Figure 3b), confirming the necessity of this A2 domain in sparsomycin biosynthesis. The exact catalytic and/or structural role of this domain is yet to be determined through further biochemical studies. In summary, we have identified the sparsomycin biosynthetic gene cluster by genome mining, targeted gene mutations and heterologous expression, which sets the stage for deciphering the chemical logic and enzymatic machinery in sparsomycin biosynthesis. Our in vitro enzymatic experiments further delineate the functions of NRPS machinery consisting of SpsQ/R/M/S in building the dipeptidyl alcohol scaffold of sparsomycin (Figure 1). This unusual NRPS machinery features the first stand-alone reductase, SpsM, which is responsible for reductive release of the tethered dipeptidyl intermediate to form a dipeptidyl alcohol, and a probable transthiolase, SpsS, that promotes transfer of an aminoacyl group between T domains on the assembly line. Further studies will focus on the functional investigation of the atypical SpsR-A2 domain and the elucidation of enzymatic pathways for formation of UAA and MTM-Cys.

METHODS

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Experimental procedures are described in detail in the Supporting Information.

ASSOCIATED CONTENT Supporting Information Details of experimental procedures are included in the Supporting Information. The DNA sequence of the sps gene cluster has been deposited into GenBank under the accession number KP861867. This information is available free of charge via the internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected]

Author Contributions §

These authors contributed equally.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We are grateful to Z. Shao (Iowa State University) for the materials and guidance of DNA assembler system. We thank C. Khosla (Stanford University) for providing Streptomyces lividans K4-114, Z. Xi (Nankai University of China) for providing sparsomycin and UAA standards, and S. Bauer (UC Berkeley) for helping with MS analysis. This work was financially supported by NSFC (31425001) (to S. L.) and the Pew Scholars Program (to W. Z.).

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Sparsomycin Biosynthesis Highlights Unusual Module Architecture

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