Class I Methyltransferase VioH Catalyzes Unusual S-Adenosyl-l

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Class I methyltransferase VioH catalyzes unusual SAM cyclization leading to 4-methylazetidinecarboxylic acid formation in vioprolide biosynthesis Fu Yan, and Rolf Müller ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00958 • Publication Date (Web): 12 Dec 2018 Downloaded from http://pubs.acs.org on December 13, 2018

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ACS Chemical Biology

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Class I methyltransferase VioH catalyzes unusual SAM cyclization leading to 4-methylazetidinecarboxylic acid formation in vioprolide biosynthesis

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Fu Yan, Rolf Müller*

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Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Centre for Infection Research and Department of Pharmacy at Saarland University, Saarland University Campus, Building E8.1, 66123 Saarbrücken, Germany.

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*E-mail: [email protected]

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ABSTRACT

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SAM-dependent methyltransferases are intensely studied since they play important roles in the methylation of biomolecules in all domains of life. In this study we describe that the methyltransferase VioH from Cysotobacter violaceus catalyzes a so far unknown cyclization of SAM to azetidine-2-carboxylic acid (AZE), which is proposed to be the precursor of the unusual 4-methylazetidinecarboxylic acid (MAZ) moiety of vioprolides. In vitro biochemical investigations reveal that SAM is converted to AZE in the presence of VioH, while MAZ is generated by coexpression of VioH and the radical SAM enzyme VioG in Myxococcus xanthus or by combination of VioH and the cell lysate of M. xanthus expressing VioG. Thus, our findings unveil a novel function of SAM-dependent methyltransferases and shed light on the biosynthetic mechanism of MAZ formation.

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INTRODUCTION

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S-adenosyl-L-methionine (SAM) dependent methyltransferases (SAM-MTs, EC 2.1.1) are omnipresent in all domains of life, performing critical roles in biosynthetic modification and functional regulation of biomolecules like nucleotides, proteins and natural products.(1–3) They usually transfer a methyl group from the ubiquitous cofactor SAM to accepting atoms such as O, C, N, S, P or even halides.(2) The versatility of SAM-MTs is also reflected by their broad variety of substrates. Up to date, more than 300 members of SAM-MTs have been classified in the Enzyme Classification system according to their substrate specificities, and over 1.8 million methyltransferase (MT) protein sequences have been deposited in the UniProt database. Based on the structural fold, MTs are currently divided into five classes, among which class I MTs represent the largest group.(3) MTs are also frequently involved in natural product biosynthetic pathways and methylation of secondary metabolites can significantly affect their bioactivities.(3) Elucidation of reaction mechanisms and rational engineering of MTs thus can contribute to the generation of novel natural product derivatives with modified bioactivities. Here, we report a novel function for a class I MT from the ACS Paragon Plus Environment

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vioprolide biosynthetic pathway, which features a previously undescribed SAM cyclization activity to yield azetidine-2-carboxylic acid (AZE).

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Vioprolides are a class of anti-fungal and cytotoxic peptolides produced by the myxobacterium Cysotobacter violaceus Cb vi35,(4) the biosynthetic pathway of which has been elucidated recently.(5) Vioprolides A and C contain an unusual 4methylazetidinecarboxylic acid (MAZ) moiety (Figure 1). In addition to vioprolides, the presence of MAZ in a natural product has only been reported for bonnevillamide A, which is a linear heptapeptide isolated from Streptomyces sp. GSL-6B recently (Figure 1).(6) The origin of the MAZ moiety in bonnevillamide is unknown, whereas feeding studies in vioprolide biosynthesis revealed that MAZ originates from methionine.(5) In the biosynthesis pathway of vioprolides the class I SAM-MT-like protein VioH and the radical SAM protein VioG were identified by gene deletion as essential for MAZ formation. VioH was proposed to cyclize the α-aminobutyric carboxylic acid moiety of SAM to form AZE, while VioG was hypothesized to methylate the resulting AZE via a radical methylation mechanism.(5) Currently, only few natural compounds are known that contain an azetidine moiety (Figure 1).(6, 7–15, 16) In plants, methionine was speculated to be the source of the AZE.(17–19) The formation of AZE in nicotianamine, a metal chelator ubiquitously produced in plants, was found to depend on SAM cyclization catalyzed by nicotianamine synthase (NAS).(20) Sequence analysis revealed a MT-like domain in the C-terminus of NAS.(21) However, the role of the MT-like domain in NAS is unclear and the catalytic mechanism for AZE formation remains elusive. The azetidine ring in okamarine B and D, indole alkaloids from Penicillium and Aspergillus species, is formed by intramolecular cyclization catalyzed by an α-ketoglutaratedependent dioxygenase OkaE,(22) while the azetidine moiety in polyoxins, nucleoside antibiotics from streptomycetes, originates from isoleucine and seems to be formed by a different mechanism.(23,24)

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Figure 1 Structures of azetidine-containing natural products.

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RESULTS AND DISCUSSION

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To investigate the mechanism of MAZ formation in vioprolide biosynthesis, we aimed to characterize the enzymatic activities of VioG and VioH in vitro. VioH was heterologously produced in Escherichia coli and purified with a yield of 4.4 mg mL-1 (Supplementary Figure 1), while no soluble VioG could be obtained from E. coli under various conditions. Purification of His6-VioG from Myxococcus xanthus failed (data not shown). A homogeneous fraction of VioH was incubated with SAM, and the resulting products were modified by Marfey’s reagent Nα-(5-Fluoro-2,4-dinitrophenyl)-D-leucinamide (D-FDLA) and analyzed by UPLC-MS. As shown in Figure 2, a compound featuring the same mass and retention time as the AZE-DLA reference compound (preparation described in Exp. Procedures) was generated by incubation of VioH and SAM. No AZE was generated in presence of VioH which was denatured at 100 oC. Detailed analysis revealed that the catalytic efficiency of VioH was remarkably low. Incubation of 50 μM VioH with 2 mM SAM at 30 oC for 16 h showed continuous production of AZE (Supplementary Figure 2). Similar results were obtained by incubation of 50 μM VioH and 50 μM SAM at 30 oC for 24 h. However, incubation of VioH and SAM in equivalent concentrations at 40 oC reavealed the decrease of reaction speed after 8 hours (Supplementary Figure 3). It seems that SAM is more stable at 30 oC and was largely degraded at 40 oC after 8 hours. We then analyzed the effects of pH, temperature and buffer on the catalytic efficiency of VioH. The activity assay was performed in a pH range from 6.5 to 12.5 and showed that the formation of AZE reached its maximum at around pH 9 and significantly decreased at pH > 11.5 (Figure 2). However, the lower production of AZE at high pH may also result from the lack of stability of SAM. Temperature curve assays revealed a steady increase of AZE production from 25 oC to 45 oC and a decline at higher temperature (Figure 2). Even at temperature as high as 50 oC, VioH still retained activity. Unexpectedly, AZE-DLA was detected in the samples incubated at high temperatures. To clarify this finding, aliquots containing only buffer and SAM were incubated at 25–100 oC for 2 h. UPLC-MS analysis detected the formation of a compound exhibiting the same mass and rentention time with AZE-DLA (Supplementary Figure 4), indicating a chemical formation of AZE from SAM which, to the best of our knowledge, has not been described in the literature. The chemically formed AZE could be detected at very low levels after reactions below 40 oC, with an increase in the production at higher temperatures. UPLC-MS/MS analysis demonstrated the VioHindependent formation of AZE at increased temperatures, especially in the range of 70– 100 oC (Supplementary Figure 5). Considering the stability of SAM, the optimal catalytic temperature for VioH is between 40 and 45 oC. To avoid possibly interfering effects from buffer components, the storage and reaction buffer of VioH was changed from Tris-NaCl to PBS, in which VioH showed a slightly higher activity (Supplementary Figure 6). The formation of AZE at concentrations below those of VioH present in the assays indicated

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a very low in vitro kcat. Due to the observed low catalytic efficiency of VioH and the issue with stability of SAM, we were not able to determine catalytic constants in vitro. Notably, AZE has been reported as a toxic non-proteinogenic amino acid, the incorporation of which can cause misfolding of proteins and inhibition of cell growth.(25–27) Hence, a low level of intracellular AZE production may protect C. violaceus from such side-effects during vioprolide biosynthesis. In addition, since VioG and VioH are proposed to work synergistically in the formation of MAZ,(5) we cannot exclude that VioG has a crucial impact on the catalytic activity of VioH, which we can currently not test in vitro.

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Figure 2 Production analysis of AZE generated in vitro. (a) scheme of Marfey’s derivatization. (b) UPLC-MS analysis AZE-DLA. The reactions were performed at 30 oC for 1 h. Similar results were obtained by using SAM from a different vendor (Supplementary Figure 7). Extracted ion chromatograms of AZE-DLA (m/z 396.14 ± 0.05 [M+H]+) is shown. (c) pH dependence of VioH activity. (d) temperature dependence of VioH activity.

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In addition to the detection of AZE, we also monitored the reaction products of SAM. As expected, the methylthioadenosine split from SAM was detected (Supplementary Figure 8). As previously reported, SAM is not stable in aqueous solution. At different pH conditions, SAM decomposes into 5’-deoxy-5’-(methylthio)adenine (MTA), adenine, S(5’-deoxy-ribosyl)-L-methionine, methionine, homoserine or homoserine lactone.(28–30) In our reactions, we detected all the hydrolytic products of SAM except for S-(5’-deoxyribosyl)-L-methionine (Supplementary Figure 8). To test if the formation of hydrolysis products from SAM mignt be catalyzed by VioH to form AZE, VioH was incubated with ACS Paragon Plus Environment

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MTA, methionine, homoserine or homoserine lactone. Subsequent UPLC-MS analysis revealed that none of these substrates could be cyclized by VioH (Supplementary Figure 9). Considering the structural similarities between SAM and its hydrolytic fragments, these degradation products from SAM may bind to VioH and inhibit the catalysis. Since SAM is degraded to methylthioadenosine naturally in aqueous solution, an SN2 reaction is proposed to take place at the C4 position of the resulting aminobutyric acid moiety, yielding homoserine, homoserine lactone or AZE (Figure 3). It is reasonable to assume that VioH deprotonates the amino group of SAM and facilitates the intramolecular cyclization between C4 and the amino group (Figure 3). Protein BLAST search against the protein databank uncovered few homologous proteins exhibiting conserved SAMbinding regions with less than 45% sequence identity to VioH, while very low sequence similarity was found among VioH and other structurally-known methyltransferases (Supplementary Figure 10). Although residues in the SAM-binding region are conserved, SAM-binding residues could vary tremendously.(1) Additionally, homology modelling of VioH is not accurate because of a lack of a good template protein structure. Thus it is difficult to predict the crucial catalytic residues in VioH based on in silico analysis. Initial attempts to (co-)crystallize VioH without and with substrate or product have not been successful so far and will be continued using a broader set of conditions in order to obtain detailed molecular insights into the catalytic mechanism.

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Figure 3 Proposed mechanism of AZE formation.

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Since soluble expression of VioG in E. coli was not achievable, we attempted to investigate the function of VioG and to produce MAZ in vivo. Three heterologous expression strains, Myxococcus xanthus::Ptet-vioG, M. xanthus::Ptet-vioH and M. xanthus::Ptet-vioGH, were generated producing either VioG, VioH, or both enzymes, respectively. After cultivation, the crude extracts of the cultures were modified by Marfey’s reagent and were analyzed by UPLC-MS. As is shown in Figure 4, MAZ could be detected in the crude extracts from M. xanthus::Ptet-vioGH in the presence of vitamin B12. Meanwhile, small amounts of AZE could also be detected. In addition, the mutant M. xanthus::Ptet-vioH produced some AZE, while neither AZE nor MAZ could be found from the mutant M. xanthus::Ptet-vioG. In our previous gene inactivation studies,



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deletion of vioG in the vio gene cluster abolished the production of vioprolides A and C without generation of AZE-containing vioprolides,(5) which may result from the low catalytic efficiency of VioH in the absence of VioG.

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Figure 4 UPLC-MS analysis of AZE-DLA and MAZ-DLA from M. xanthus mutants. Extracted ion chromatograms of AZE-DLA (m/z 396.14 ± 0.05 [M+H]+, 13.22 min), MAZDLA (m/z 410.20 ± 0.05 [M+H]+, 14.40 min) and D-Pro-DLA (m/z 410.20 ± 0.05 [M+H]+, 14.15 min) are shown. A, M. xanthus negative control; B, M. xanthus::Ptet-vioG; C, M. xanthus::Ptet-vioH; D, M. xanthus::Ptet-vioGH without feeding of vitamin B12; E, M. xanthus::Ptet-vioGH fed with vitamin B12; F, MAZ-DLA reference; G, AZE-DLA reference; H, D-Pro-DLA reference. The AZE-DLA peaks in D and E were magnified by 10 fold above the line of the chromatogram. The peaks of D-Pro-DLA are shown because of their isobaric mass to MAZ-DLA. The retention time of L-Pro-DLA is out of the time frame shown (retention time 15.84 min under these conditions).

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After showing the formation of MAZ in the heterologous M. xanthus strains, we tested an alternative in vivo – in vitro combined approach to reconstitute MAZ production. The M. xanthus::Ptet-vioG cell lysates were incubated with purified recombinant VioH, and the products were modified by D-FDLA and analyzed by UPLC-MS. As shown in Figure 5, the formation of MAZ could only be detected in the presence of VioH in the cell lysate. Notably, in the absence of VioH, MAZ could not be generated by incubation of M. xanthus::Ptet-vioG cell lysates with AZE. It seems that free AZE cannot be methylated by VioG. It is likely that VioG can only accept modified or protein-bound AZE, hence an interaction of VioG and VioH might be essential for the generation of MAZ. In previous in vivo experiments, vitamin B12 was found essential for the formation of MAZ, and thus it was added to the cultivation medium of M. xanthus::Ptet-vioG. Supplementing vitamin B12 to cell lysates did not show an effect on the production of MAZ (Figure 5). Interestingly, the variation of SAM concentrations also showed no significant impact on the formation of MAZ. Even if no further SAM was added to the reaction, MAZ was still


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formed (Figure 5, D and F), indicating that the SAM present in the cell lysate seems to be sufficient for the observed product formation. The overall low production yield of MAZ in different reaction systems indicated limited catalytic activity of VioG, which may result from the residual oxygen in the reactions. Since the [4Fe–4S] cluster in radical SAM enzymes is sensitive to oxygen,(31) incubation of M. xanthus::Ptet-vioG cell lysates and VioH under strict anaerobic conditions may improve the production of MAZ. In addition, it cannot be excluded that SAM is methylated by VioG before being cyclized by VioH. However, no products related to methyl-SAM could be detected in the incubation of the cell lysate with SAM. Due to the difficulties in obtaining soluble VioG and methyl-SAM, the mechanism of MAZ formation cannot be elucidated in detail at present.

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Figure 5 UPLC-MS analysis of MAZ-DLA generated by whole cell lysate and VioH. Extracted ion chromatograms of MAZ-DLA and D-proline (m/z 410.20 ± 0.05 [M+H]+) are shown. A, M.xanthus DK1622 (∆mchA) + VioH + SAM + VB12; B, M.xanthus::Ptet-vioG + AZE + SAM without VioH; C, M.xanthus::Ptet-vioG + VioH + SAM + VB12; D, M.xanthus::Ptet-vioG + VioH + VB12 without SAM; E, M.xanthus::Ptet-vioG + VioH + SAM without VB12; F, M.xanthus::Ptet-vioG + VioH + AZE without SAM; G, M.xanthus::Ptet-vioG + VioH + SAM +VB12 without ferric ion; H, M.xanthus::Ptet-vioG + VioH + SAM without VB12 and ferric ion; I, M.xanthus::Ptet-vioG + VioH + SAM + VB12 + (NH4)2Fe(SO4)2; J, M.xanthus::Ptet-vioG + VioH + SAM + VB12 + FeCl2; K, MAZ-DLA reference; L, D-Pro-DLA reference. FeCl3 was added to A – F. Detailed reaction conditions are provided in Supplementary Table S1.

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In summary, we reconstituted azetidine-2-carboxylic acid formation by an unusual class I SAM-dependent methyltransferase VioH in vitro. This study thus provides the first example for a new function of class I SAM-MT: the intramolecular cyclization of SAM. The reconstitution of AZE and MAZ via in vivo genetic engineering and in vitro biochemical reactions sets the stage for the detailed elucidation of the biosynthetic mechanism of these unusual amino acids and provides opportunities to generate novel natural products by applying them to combinatorial biosynthesis. Considering that nicotianamine synthase in plants also contains a MT-like domain, a SAM-cyclizing function of MTs could be more widespread among organisms from different kingdoms than currently expected.

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METHODS

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Strains and cultivation conditions

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Strains used in this study are listed in Supplementary Table S2. E. coli strains were cultivated in LB broth for routine propagation. M. xanthus DK1622 was cultivated in CTT medium. Kanamycin (50 μg mL-1), chloramphenicol (20 μg mL-1) or oxytetracycline (5 μg mL-1) was added to culture when needed.

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Construction of expression plasmids

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Primer sequences are listed in Supplementary Table S2. The genes vioG and vioH were amplified from p15A-Ptet-vio using the primer pairs NheI-orf5-5 / EcoRI-orf5-3 and NheIorf6-5 / EcoRI-orf6-3, and was inserted between NheI and EcoRI sites downstream of the TEV-protease cleavage site on the expression vector pET28b-sumo-tev, resulting His6-Sumo-TEV-VioG and His6-Sumo-TEV-VioH expression vectors pET-STVioG and pET-STVioH, respectively. The open reading frames were validated by Sangersequencing.

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Generation of M. xanthus mutants

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The VioG, VioH and VioGH expression constructs p15A-Ptet-vioG, p15A-Ptet-vioH and p15A-Ptet-vioGH were generated by engineering p15A-Ptet-vio. The cm-ccdB cassette was amplified from p15A-ccdB-Cm using primers tetR-cmR-XmaJI-5 and Orf3-ccdB-3, and was transferred into recombinase induced E. coli GBred-gyrA462::p15A-Ptet-vio. The vio gene cluster of p15A-Ptet-vio was then replaced by cmR-ccdB via Red/ET recombination,(32) the resulting plasmid p15A-Ptet-vioorfs-cmccdB was further hydrolyzed by XhoI and cyclized by T4 DNA ligase, yielding p15A-Ptet-cmccdB. The genes vioGH were amplified from p15A-Ptet-vio using the primers Ptet-orf56-5 and Ptetorf56-3 and, the resulting fragment was constructed to p15A-Ptet-cmccdB by Red/ET recombination, yielding p15A-Ptet-vioGH. The fragment cmR-delorf6 was amplified from ACS Paragon Plus Environment

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p15A-Ptet-cmccdB using primers orf5-cmR-XmaJI-5 and cmR-XmaJI-MluI-3, and was transferred into recombinase proficient E. coli GBred::p15A-Ptet-vioGH. The vioH of p15A-Ptet-vioGH was then replaced by cmR gene via Red/ET recombination. The resulting plasmid p15A-Ptet-vioG-cmR was hydrolyzed by XmaJI and cyclized by T4 DNA ligase, yielding p15A-Ptet-vioG. The fragment tetR-Ptet was amplified from p15APtet-vioGH using primers tetR-Ptet-XmaJI-5 and Ptet-orf6-I, and vioH was amplified using primers Ptet-orf6-II and orf6-MluI-3. The two fragments were ligated by overlap extension PCR to generate tetR-Ptet-vioH cassette. The tetR-Ptet-vioG cassette of p15A-Ptet-vioG was replaced by tetR-Ptet-vioH cassette at XmaJI and MluI sites, yielding p15A-Ptet-vioH. The resulting plasmids p15A-Ptet-vioG, p15A-Ptet-vioH and p15A-Ptet-vioGH were transferred into Myxococcus xanthus DK1622 (∆mchA), and the Ptet-vioG-, Ptet-vioH- or Ptet-vioGH-containing cassette was integrated to the chromosome via transposition.

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Protein expression and purification

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The VioH expression vector pET-STVioH was transferred into E. coli Rosetta (DE3). The resulting strains were cultivated in LB broth at 37 oC overnight. Proportion of 1% (v/v) overnight cultures was inoculated into ZYM-5052 auto-induction medium. After cultivation at 37 oC for 30 min, the cultures were moved to 16 oC and cultivated for 2 days. The cells were collected and stored at -80 oC before protein purification. The cells were suspended in 200 mL wash buffer (20 mM Tris, 150 mM NaCl, 20 mM imidazole, pH 8.0) and were lysed by cell disruptor (Microfluidics Corp.). A tablet of cOmplete™, EDTA-free Protease Inhibitor Cocktail (Roche) was added during cell lysis. Cell debris was removed by centrifugation at 19000 rpm and 4 oC for 40 min. The supernatant was loaded to HisTrap HP 5 mL column connected to AKTA avant and His6-Sumo-TEV-VioH was eluted by a gradient increase of imidazole concentration. The protein was desalted on HiPrep Desalting column (20 mM Tris, pH 8.0), and was then purified with HiTrap Q HP 5 mL column connected to AKTA purifier (gradient concentration of NaCl from 0– 100%). The His6-Sumo-tag was cleaved by incubation with TEV-protease at 4 oC overnight, and was removed by incubation with Ni-NTA agarose resin (Qiagen) and loading to gravity column. The eluents containing tag-free VioH were collected and buffer exchanged using HiPrep Desalting column (20 mM Tris, 200 mM NaCl, 10% glycerol, pH 8.0). The homogenous VioH were concentrated using centrifugal filter (Millipore, 10 kDa cut off), snap frozen in liquid nitrogen and stored at -80 oC.

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In vitro biochemical assays

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To measure the activity of VioH, 50 μL reaction containing 300 μM protein, 8 mM MgCl2, 2 mM SAM (Sigma or MedChem Express) and buffer (20 mM Tris, 200 mM NaCl, pH 8.0) was incubated at 30 oC for 1 hour. The VioH heated at 100 oC for 5 min was set as negative control. Large molecules were removed by centrifugal filter (Millipore, 10 kDa cut off), and the flow through was adjusted to 50 μL with buffer and modified by Marfey’s ACS Paragon Plus Environment


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derivatization (see below). To measure the impact of temperature, 50 μL reaction containing 10 μM protein and 2 mM SAM was incubated at 25–100 oC for 2 hours. The test of pH effects was performed in 50 μL reaction containing 10 μM protein, 2 mM SAM and buffer (20 mM Tris, 200 mM NaCl, pH 6.5–13), and was incubated at 40 oC for 2 hours. The reaction buffer was adjusted to pH 6.5–13 using HCl or NaOH. Since the pH of VioH storage buffer has an effect on the pH of reaction buffer, the pH in the measurements was initially determined by measuring mixtures of storage buffer and reaction buffers at larger scale using a pH meter, and the pH of actual reaction systems was monitored by pH strips. To test effects of buffer component to the activity of VioH, the storage buffer was changed to phosphate buffered saline (PBS, pH 8.0) using Hiprep Desalting column. The 50 μL reactions containing 10 μM protein, 2 mM SAM and PBS buffer (pH 8.0) were performed at 30–90 oC for 2 hours. The reactions were directly modified by Mafey’s derivatization (see below).

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Derivatization of AZE and LC-MS measurement

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The AZE or MAZ was analyzed by Marfey’s assays.(33) The volume of 20 μL 1M NaHCO3 and 20 μL Marfey’s reagent Nα-(5-Fluoro-2.4-dinitrophenyl)-D-leucinamide (DFDLA, 1% in acetone) were added to 50 μL samples. After incubation at 40 oC with shaking (700 rpm) for 1h, the reaction was stopped by adding 10 μL 2 M HCl and 300 μL acetonitrile. UPLC-MS measurements of the modified AZE (AZE-DLA) were performed on an Dionex Ultimate 3000 RSLC system (Thermo Scientific) using a BEH C18, 100 x 2.1 mm, 1.7 μm dp column (Waters, Germany) coupled to a Bruker Maxis 4G mass spectrometer. Separation of 1 μL sample was achieved by a linear gradient from (A) H2O to (B) methanol at a flow rate of 600 μL min-1 and 45 oC: 0–1 min, 5–10% B; 1–15 min, 10–35% B; 15–22 min, 35–55% B; 22–25 min, 55–80% B; 25–26 min, 80% B, 26– 26.5 min, 80–5% B, 26.5–31 min, 5% B. Full scan mass spectra were acquired in a range from 100–1000 m/z. Quantification of AZE-DLA was carried out on Dionex Ultimate 3000 RSLC system (Thermo Scientific) coupled to a Bruker amaZon speed mass spectrometer. Sulfamethizole (MW 270.333 g mol-1) was added to the samples and served as internal reference. AZE referential compound (Sigma Aldrich) was modified with D-FDLA and diluted to 0.01–5 μM. Calibration curve was obtained by measurements of AZE-DLA references. Separation of 1 μL sample was achieved on a ACQUITY UPLC CSHTM Fluoro-Phenyl column (100 x 2.1 mm, 1.7 μm dp, Waters, Germany) by a linear gradient from (A) H2O to (B) acetonitrile at a flow rate of 600 μL min-1 and 45 oC: 0–0.5 min, 5% B; 0.5–4 min, 5–25% B; 4–18 min, 25–33% B; 18–19.5 min, 33–95% B; 19.5–20.5 min, 95% B, 20.5–20.8 min, 95–5% B, 20.8–21.5 min, 5% B. Full scan mass spectra were acquired in a range from 50–800 m/z. The areas of MS/MS fragments of sulfamethizole (155.85 ± 0.3, [M+H]+) and AZE-DLA (351.08 ± 0.3, [M+H]+) (Supplementary Figure 11) were integrated and analyzed using the software Bruker Compass QuantAnalysis Version 2.2.

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In vivo production of MAZ

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The mutants M. xanthus::Ptet-vioG, M. xanthus::Ptet-vioH and M. xanthus::Ptet-vioGH were cultivated in 50 mL CTT medium (casitone 10 g L-1; 10 mM Tris∙HCl, pH 7.6; 1 mM K2HPO4, pH 7.6; 8 mM MgSO4) at 30 oC for 4.5 days. The portion of 1% Amberlite XAD16 resin was added to the cultures. The compounds were extracted with methanol and evaporated in a vacuum system. The dryness was suspended in 50 μL ddH2O and modified with Marfey’s reagent as mentioned above. Authentic L-MAZ (synthesized previously),(5) L-AZE (Sigma), and D-proline were modified with D-FDLA and served as references. The production of MAZ was measured on Bruker Maxis 4G mass spectrometer as mentioned above.

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Production of MAZ from whole cell extracts

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M. xanthus DK1622 (∆mchA) and M. xanthus::Ptet-vioG were cultivated in CTT medium containing 2 mg L-1 vitamin B12 at 30 oC for 2 days. The production of MAZ using whole cell extracts of M. xanthus::Ptet-vioG and the purified VioH was achieved in 250 μL PCR tube. A total of 250 μL reaction system contains ~30 mg M. xanthus::Ptet-vioG cells, 50 μM VioH, 1 mM vitamin B12, 8 mM MgCl2, 0.5 mM FeCl3 / (NH4)2Fe(SO4)2 / FeCl2, 0.5 mM Na2S and 2 mM sodiumdithionate. The tubes were flushed under nitrogen immediately after adding 0.2 mg mL-1 lysozyme and CelLytic B cell lysis reagent (Sigma). The reaction was performed at room temperature for 5 hours. The particles and large molecules were removed by centrifugal filter (Millipore, 10 kDa cut off), and 50 μL eluent was modified by Marfey’s reagent. The production of MAZ was measured by UPLC-MS as mentioned above.

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ACKNOWLEDGEMENT

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We thank M. Miethke for helpful suggestions regarding the manuscript and D. Sauer for LC-MS measurements.

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FUNDING SOURCES

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This work was partially funded by the Deutsche Forschungsgemeinschaft (MU 1254/142).

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SUPPORTING INFORMATION

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Supporting Information Available: This material is available free of charge via the Internet.

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SDS-PAGE and mass spectra of VioH, LC-MS analysis, strains and primers.



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618x303mm (96 x 96 DPI)


Class I Methyltransferase VioH Catalyzes Unusual S-Adenosyl-l

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