Mutations of Residues in Pocket P1 of a Cyclodipeptide Synthase

Article Cite This: J. Nat. Prod. 2017, 80, 2917-2922

pubs.acs.org/jnp

Mutations of Residues in Pocket P1 of a Cyclodipeptide Synthase Strongly Increase Product Formation Kirsten Brockmeyer and Shu-Ming Li* Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Robert-Koch-Straße 4, 35037 Marburg, Germany S Supporting Information *

ABSTRACT: Expression of a cyclodipeptide synthase gene from Nocardiopsis prasina (CDPS-Np) in Escherichia coli resulted in the formation of cyclo-(L-Tyr-L-Tyr) (1) as the minor and cyclo-(L-Tyr-L-Phe) (2) as the major products. Sitedirected mutagenesis revealed a strong influence on product accumulation of the amino acid residues in pocket P1. An 8fold increase in product formation for 1 and 10-fold for 2 were detected in the double mutant T82V_Y196F compared with the wild type.

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dipeptides; cyclo-(L-Tyr-L-Tyr) was detected as the main product (92%) of the CDPS from Streptomyces venezuelae9 and Rv2275 from Mycobacterium tuberculosis produces 91% cyclo-(L-Tyr-L-Tyr) and 6% cyclo-(L-Tyr-L-Phe).11 Based on crystal structures and mutagenesis experiments, CDPSs are proposed to contain two binding pockets, P1 and P2, with defined amino acid motifs for the two aminoacyltRNAs (Table S1), respectively.9,16,17 It was postulated that P1 is more specific for binding the first aminoacyl-tRNA, usually found as the common amino acid of the cyclodipeptide mixtures.17 The wider binding pocket P2 is responsible for the less specific binding of the second and often variable aminoacyltRNA.16 Based on previous studies, even small changes within the sequence motif of P1 can lead to accommodation of different aminoacyl-tRNAs.9 For example, Rv2275 mentioned above and Amir_4627 from Actinosynnema mirum differ in only two amino acid residues within the P1 motif, but produce cyclo(L-Tyr-L-Tyr) and cyclo-(L-Trp-L-Trp) as their main products, respectively (Table S1).11,12 A previous study showed that mutation of just one amino acid (L200N) within P1 of AlbC led to a shift in production from cyclo-(L-Phe-L-Leu) to cyclo-(LTyr-L-Leu).18 However, the influence of the residues in P1 on the enzyme activity for the same substrates has not been reported previously. In the course of our investigations on DKP-forming enzymes,19,20 one CDPS candidate, WP_017544375.1 from Nocardiopsis prasina (N. prasina), named CDPS-Np in this study, attracted our attention. The Gram-positive bacterium N. prasina is known to produce polyketides, e.g., kalafungin as well

econdary metabolites with a 2,5-diketopiperazine (DKP) skeleton frequently exhibit diverse biological and pharmacological activities1,2 and represent promising drug candidates.3−5 These include phenylalanine- and tyrosine-containing DKP derivatives (Chart 1). For example, plinabulin acts as a potent microtubule inhibitor and reached phase III clinical trial (NCT02504489, ClinicalTrials.gov).6 While albonoursin exhibits antibiotic activity against Bacillus spp., its paramethoxylated derivative is a cytotoxic agent against NIH-3T3 and HepG2 cells.1 XR334 can reverse the inhibitory effects of plasminogen activator inhibitor-1 (PAI-1).1 The formation of the DKP skeleton is catalyzed either by two-modular nonribosomal peptide synthetases (NRPSs) or by cyclodipeptide synthases (CDPSs).7,8 The majority of CDPSs comprise 200−300 amino acids. They hijack aminoacyl-tRNAs from the ribosomal peptide biosynthesis and use them as substrates for the formation of two successive peptide bonds in an ATP-independent manner.8−13 By the end of July 2016, about 450 putative CDPS genes, most in actinobacteria and a few in eukarya including fungi, protozoa, and animals, were retrieved by searching the National Center for Biotechnology Information (NCBI) protein database.14 Sixty-four of these genes have been proven to be responsible for the formation of different DKPs.9,14,15 AlbC was the first described CDPS from Streptomyces noursei and produces phenylalanine-containing cyclodipeptides such as cyclo-(L-Phe-L-Leu), cyclo-(L-Phe-LPhe), cyclo-(L-Phe-L-Tyr), and cyclo-(L-Phe-L-Met), with the first one as the predominant product.10,11 The CDPS Ndas_1148 from Nocardiopsis dassonvillei produces cyclo-(LPhe-L-Tyr), cyclo-(L-Phe-L-Phe), and cyclo-(L-Phe-L-Leu) as its major and cyclo-(L-Phe-L-Ala), cyclo-(L-Phe-L-Met), cyclo-(LTyr-L-Leu), and cyclo-(L-Tyr-L-Tyr) as its minor products.13 Two CDPSs produce predominantly tyrosine-containing cyclo© 2017 American Chemical Society and American Society of Pharmacognosy

Received: May 17, 2017 Published: October 24, 2017 2917

DOI: 10.1021/acs.jnatprod.7b00430 J. Nat. Prod. 2017, 80, 2917−2922

Journal of Natural Products

Article

as nocapyrone E,21,22 but until now neither DKPs or their derivatives were reported. According to the classification,9 WP_017544375.1, consisting of 242 amino acids, belongs to the NYH superfamily containing the catalytic residues Asn40, Tyr178, and His203 (AlbC numbering) (Figure S1). Comparing its sequence logo in pockets P1 and P2 (Table S1) with those of several known CDPSs (Figure S1) revealed that, with the exception of Tyr196, CDPS-Np shares identical amino acids in other positions with one or more CDPSs in pocket P1 (Table S1). According to the proposed logo alignment, CDPS-Np was proposed to be responsible for the formation of phenylalanine-containing DKPs.9

shown in Figure S2A, peak 1 shares the same MS2 fragment pattern23 with an authentic cyclo-(L-Tyr-L-Tyr) standard at m/z 299.14, 254.12, 221.09, and 136.08, proving unequivocally the identity of peak 1 as cyclo-(L-Tyr-L-Tyr). Peak 2 was eluted at 15.4 min (Figures 1 and 2) and has a [M + H]+ ion at m/z 311.1378, 16 units smaller than that of 1, indicating the presence of cyclo-(L-Tyr-L-Phe) with a calculated [M + H]+ ion at m/z 311.1390. Indeed, 2 has the same retention time, UV spectrum, and MS2 fragments (283.14, 238.12, 205.10, 136.08, and 120.08, Figure S2B) as an authentic cyclo-(L-Tyr-L-Phe) standard. In addition, two products, 3 and 4, with [M + H]+ ions at 295.1081 (C14H19N2O3S) and 277.1529 (C15H21N2O3), respectively, were also detected with higher intensities than in the negative control in extracted ion chromatograms (EICs, Figure 3) and indicated the presence of cyclo-(L-Tyr-L-Met) (3) and cyclo-(L-Tyr-L-Leu) or cyclo-(L-Tyr-L-Ile) (4). However, their yields were too low to be detected by UV absorption, so that a quantification was not possible. All the results provided evidence that CDPS-Np from N. prasina functions as a tyrosine-containing DKP-forming enzyme. The productivity of the cells harboring pKB25 was calculated to be 0.19 mg/L for 1 and 1.29 mg/L for 2, i.e., in a ratio of 1:6.8 (Table 1). Tyr196 in P1 of CDPS-Np (Table S1) seems to be involved in the binding of tyrosyl-tRNA. In comparison, AlbC and Ndas_1148 both have a leucine in this position, while other CDPSs from different Nocardiopsis strains share a phenylalanine (Figure S1). We attempted to change the substrate specificity of CDPS-Np by site-directed mutagenesis24 and created two constructs, pKB106 for the mutant CDPS-Np_Y196L and pKB108 for CDPS-Np_Y196F (see Experimental Section). The cultures harboring pKB25, pKB106, and pKB108 were treated as mentioned above, and the accumulation of the secondary metabolites was monitored by LC-MS analysis. In the case of CDPS-Np_Y196L, the formation of both 1 and 2 decreased to 84% of those of CDPS-Np. The yields of 1 and 2 in the mutant CDPS-Np_Y196F increased to 3.1- and 2.4-fold of those of CDPS-Np, respectively (Table 1 and Figure 2). This indicated that product yields can be influenced by alteration of the binding site without significant change of the substrate specificity. These results encouraged us to mutate Thr82 of CDPS-NP, a strongly variable position in CDPSs (Table S1 and Figure S1). Analysis of the secondary metabolite production revealed that the formation of 1 and 2 in the mutant CDPS-Np_T82A (pKB101) was reduced to 68% and 46% of those of CDPS-NP, respectively. In contrast, mutation of Thr82 to valine showed a positive effect regarding the enzyme activity. In comparison to those of CDPS-NP, a 1.4-fold product yield for 1 and 2.2-fold for 2 were calculated in the culture with CDPS-Np_T82V (pKB102) (Table 1 and Figure 2). To ensure that the observed differences in product yields do not arise from differences in expression levels, the protein crude extracts were separated by SDS-PAGE and visualized by Coomassie staining (Figure 4A). An additional band each with the expected size of about 28 kDa was detected for CDPS-Np and its mutants, compared to the control with the empty vector. The identity of the His6-tagged proteins was confirmed by Western blot analysis (Figure 4B). The intensities of CDPSNp and the four single mutants are comparable, proving that the observed product yields are results of different catalytic activities of Tyr196 and Thr82 mutants.

Chart 1

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RESULTS AND DISCUSSION To prove its function, the coding sequence of CDPS-Np was amplified from genomic DNA of N. prasina NRRL B-16235 by using the primers Np002-for and Np002-rev (see Experimental Section) and cloned into pQE-60, resulting in the expression construct pKB25. CDPS-Np was subsequently overproduced in E. coli M15 [pREP4] cells by induction with 1 mM isopropyl-βD-thiogalactopyranoside. Culture with pQE-60 was used as the control. The cultures were extracted with ethyl acetate and subsequently analyzed on LC-MS (see Experimental Section). In comparison to that of the control, the HPLC chromatogram of the extract with pKB25 showed two additional peaks, 1 and 2, both with UV and extracted ion detection (Figures 1 and 2). Peak 1, with a retention time of 9.7 min, has a [M + H]+ ion at m/z 327.1317, which corresponds very well to the calculated [M + H]+ ion at m/z 327.1339 of cyclo-(L-Tyr-L-Tyr). As Table 1. Product Yields of CDPS-Np and Its Mutants

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Figure 1. HPLC chromatograms for detection of product formation in cultures with CDPS-Np and CDPS-Np_T82V_Y196F.

Figure 2. LC-MS analysis of the 1 and 2 formation by CDPS-Np and its mutants. Illustrated are the extracted positive-ion chromatograms (EIC) at m/z 327.1339 ± 0.005 ([M + H]+ of 1) and 311.1390 ± 0.005 ([M + H]+ of 2).

As a logical consequence, we created four double mutants on Tyr196 and Thr82, i.e., T82A_Y196L (pKB113), T82A_Y196F (pKB115), T82V_Y196L (pKB117), and T82V_Y196F (pKB119). SDS-PAGE analysis of the protein crude extracts revealed a strong influence of double mutation on the gene expression (Figure 4A). pKB119 (T82V_Y196F) has a comparable expression level to pKB25 (CDPS-Np), while the concentrations of T82A_Y196F and T82V_Y196L are about 50% and 30% of that of CDPS-Np, respectively. In the case of

T82A_Y196L, the protein yield is very low (Figure 4A). A comparison of the product yield in this mutant with that of CDPS-Np is therefore not possible. Double mutants T82A_Y196F and T82V_Y196L containing one positive and one negative mutation increased the product formation when compared with the nonmutated CDPS-Np, although their concentrations in the protein crude extracts are lower than that of the wild type. T82V_Y196L produces 0.38 mg/L 1 and 4.15 mg/L 2, which is 2.0-fold for 1 and 3.2-fold for 2 when 2919



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Figure 3. LC-MS analysis of the formation of 3 and 4 by CDPS-Np and its mutants. The extracted positive-ion chromatograms (EIC) at m/z 295.1111 ± 0.0025 ([M + H]+ of 3) and 277.1547 ± 0.0025 ([M + H]+ of 4) are illustrated.

importance of experimental proof of gene functions. Previous attempts to change the amino acids within the catalytic and conserved residues resulted in decreased product formation.18 Mutations on Thr82 and Tyr196 of CDPS-Np described in this study led to a significant improvement in product formation. These results increase the scope of synthetic biology and could also be interesting for genetic manipulation of other CDPSs.

compared with CDPS-Np. Concentrations of 0.40 mg/L 1 and 2.01 mg/L 2 are detected in the culture of T82A_Y196F. Taking the protein concentrations into consideration, the product yield reaches 4.2-fold for 1 and 3.1-fold for 2 compared with that of the wild type. As expected, the combination of two positive mutations in T82V_Y196F led to a synergy effect and an 8-fold production of 1 and 10-fold of 2 (Table 1 and Figure 2). Inspection of the product yields in CDPS-NP and its mutants (Table 1) indicated that mutation on Thr82 and Tyr196 changed the ratio of 1 and 2, from 1:11.5 to 1:3.5. With the enhanced activities of the mutants obtained, the product yields of 3 and 4 were also increased (Figure 3) in T82V, T82V_Y196L, and T82V_Y196F. In EIC of the culture with T82V_Y196F, for example, intensities of 3 and 4 were increased 11.8 and 3.7 times, respectively. MS2 fragments of 3 at m/z 247.11 and 4 at 249.16, 171.11, and 136.07 provided additional evidence for their identities as cyclo-(L-Tyr-L-Met) and cyclo-(L-Tyr-L-Leu) or cyclo-(L-Tyr-L-Ile), respectively. However, their product yields are still too low to be quantified and compared with those of 1 and 2. A time course for the production of 1 and 2 in cultures harboring pKB25 (CDPSNP) and pKB119 (CDPS-NP_T82V_Y196F) is shown in Figure 5. In this study, we identified WP_017544375.1 from N. prasina to be a tyrosine-containing cyclodipeptide synthase with cyclo(L-Tyr-L-Phe) as the main and cyclo-(L-Tyr-L-Tyr), cyclo-(L-TyrL-Met), and cyclo-(L-Tyr-L-Leu) or cyclo-(L-Tyr-L-Ile) as minor products, which differs from the predicted phenylalaninecontaining CDPs by bioinformatic analysis.9 This indicates the

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EXPERIMENTAL SECTION

General Experimental Procedures. The authentic standards cyclo-(L-Tyr-L-Tyr) and cyclo-(L-Tyr-L-Phe) were synthesized as described previously.13,25,26 High-resolution mass spectrometric data were generated by using a micrOTOF-Q III mass spectrometer with an ESI source (Bruker Daltonics, Bremen, Germany) connected to an Agilent HPLC series 1260 (Böblingen, Germany), which is equipped with a photodiode array detector. HPLC separation was carried out on a Multospher 120 RP 18 column (2 × 250 mm, 5 μm) from CSChromatographie Service GmbH (Langerwehe, Germany). Biological Material. N. prasina NRRL B-16235 was kindly provided by ARS Culture Collection (NRRL) and cultivated in a 250 mL cylindrical flask containing 50 mL of GYM media (glucose: 4.0 g L−1, yeast extract: 4.0 g L−1, and malt extract: 10.0 g L−1) at 28 °C and 180 rpm for 7 days in the dark. DNA Isolation. For isolation of genomic DNA from N. prasina NRRL B-16235, cells of the 7-day-old culture were collected and washed with TSE buffer (25 mM Tris-HCl, 10% saccharose, 25 mM ethylenediaminetetraacetic acid, pH 8.0). Genomic DNA was isolated according to a method described previously.27 PCR Amplification of the Coding Sequence of CDPS-Np. The nucleotide sequence encoding CDPS-Np (NCBI accession number WP_017544375.1) consisting of 729 bp (bps 32019−32747, 2920



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Figure 4. Analysis of CDPS-Np and its mutants. (A) The protein crude extracts were separated on a 15% SDS polyacrylamide gel and stained with Coomassie Brilliant Blue G-250. (B) The separated proteins were transferred onto a polyvinylidene difluoride membrane, and the proteins of interest were detected with antihistidine antibody as described in the Experimental Section. The red arrows indicate the overproduced histidinetagged CDPS-Np and its mutants with a calculated size of 28 kDa. and Np002-rev (5′-CGTTAGATCTCCTCGGCCTCAGGACGAG3′) were used. Bold letters in Np002-for and Np002-rev represent mutations inserted compared to the native sequence in order to create the two underlined restriction sites NcoI and BglII. The thermal profile of PCR amplification was created according to the manufacturer’s instructions, using an annealing temperature of 55 °C and an elongation time of 1 min. Gene Cloning. Standard protocols for DNA isolation and manipulation in E. coli were performed as described previously.28 The amplified DNA fragment encoding CDPS-Np was cloned into pGEM-T Easy (Promega), resulting in pKB20, which was sequenced by SEQLAB (Göttingen, Germany). In comparison to the annotated sequence in the NCBI database, thymine instead of guanine was detected at position 221 in all eight sequenced plasmids, resulting in the change of Cys74 to Phe in the amino acid sequence. The insert was then released by restriction with NcoI/BglII and recloned into the pQE-60 vector (QIAGEN), resulting in the construct pKB25 for expression in E. coli. Site-Directed Mutagenesis. The mutants of CDPS-Np were created according to a site-directed mutagenesis protocol as described previously.24 In order to create the mutants T82A, T82V, Y196L, and Y196F of CDPS-Np, the construct pKB25 was used as template. The primers Np010-for (5′-CTCTACSYGGACCTGCACCTCGACACCATG-3′) and Np010-rev (5′-GGTCCRSGTAGAGCAGGTCCACTGCGGC-3′) were used to create the two constructs pKB101 (T82A) and pKB102 (T82V), whereas Np011for (5′-CTGCCCYTCCTGCTCAGCACCCCCGAGG-3′) and

Figure 5. Time-dependent formation of 1 and 2 by CDPS-Np and CDPS-Np_T82V_Y196F. NZ_ANAE01000117) was amplified by PCR using the Expand High FidelityPLUS PCR System from Roche Diagnostics GmbH (Mannheim, Germany) on a MyCycler thermal cycler from Bio-Rad Laboratories GmbH (München, Germany). For this purpose, the two primers Np002-for (5′-AGCCCCATGGCGTTTCAGCCTGAGACAA-3′) 2921



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Np011-rev (5′-GCAGGAYGGGCAGTTCGGCGCGCAGGTAG-3′) were used to create pKB106 (Y196L) and pKB108 (Y196F). To generate the double mutants T82A_Y196L and T82A_Y196F as well as T82V_Y196L and T82V_Y196F, pKB101 and pKB102 were used as template, respectively. The primers Np011-for and Np011-rev were used in order to mutate Y196F. Np013-for (5′-CTGCCCCTCCTGCTCAGCACCCCCGAGG-3′) and Np013-rev (5′GCAGGAGGGGCAGTTCGGCGCGCAGGTAG-3′) are primers for mutation of Y196L. The conditions of PCR amplification were created as mentioned above, using an annealing temperature of 60 °C and an elongation time of 4 min. Overproduction of CDPS-Np and Its Mutants in E. coli. For overproduction of CDPS-Np and its mutants, 50 mL of LB media each inoculated with E. coli M15 [pREP4] containing one of the plasmids were cultivated at 37 °C and 230 rpm to an absorption of 0.6 at 600 nm. Gene expression was induced by using 1 mM isopropyl-β-Dthiogalactopyranoside. After incubation for a further 16 h at 37 °C and 230 rpm, 1 mL of culture was extracted with ethyl acetate (volume 1:1) and afterward resolved in 40 μL of methanol for LC-MS analysis. Detection of CDPS-Np and Its Mutants in E. coli. The protein overproduction was analyzed by SDS-PAGE according to the method by Laemmli.29 A 1 mL amount of overnight cultures each was centrifuged, and the supernatant was discarded. The pellets were suspended in the same volume of sample buffer (60 μl). The proteins in crude extracts were separated on a 15% SDS polyacrylamide gel and stained with Coomassie Brilliant Blue G-250 afterward. For Western blot analysis, the proteins were separated on a 15% SDS polyacrylamide gel and transferred to a polyvinylidene difluoride membrane using the Criterion Blotter (BioRad). The His6-tagged proteins were detected with primary (anti-histidine-tagged protein mouse mAb, Calbiochem) and secondary antibody (anti-mouse IgG alkaline phosphatase antibody, Sigma). Nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate were used for visualization. Product Analysis on LC-MS. For analysis of the products, a linear gradient of 5−100% acetonitrile in water, each with 0.1% formic acid, in 40 min and a flow rate at 0.25 mL/min was used. The column was washed afterward with 100% acetonitrile containing 0.1% formic acid for 5 min and equilibrated with 5% acetonitrile in water containing 0.1% formic acid for 5 min. The parameters for the spectrometer were adjusted to electrospray positive-ion mode for ionization, a capillary voltage of 4.5 kV, and a collision energy of 8.0 eV.

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Deutsche Forschungsgemeinschaft (INST 160/620-1 to S.M.L.).

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00430. Multiple sequence alignments of selected CDPSs and amino acid residues in the two binding pockets; MS2 analysis of 1 and 2 (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +49-6421-28-22461/25365. E-mail: shuming.li@ staff.uni-marburg.de. ORCID

Shu-Ming Li: 0000-0003-4583-2655 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS N. prasina NRRL B-16235 was kindly provided by ARS Culture Collection (NRRL). We thank L. Ludwig-Radtke and R. Kraut from Philipps-Universität Marburg for synthesis of cyclo-(L-TyrL-Phe) and taking mass spectra, respectively. The Bruker microTOF QIII mass spectrometer was funded by the 2922


Mutations of Residues in Pocket P1 of a Cyclodipeptide Synthase

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