In vitro generation of FR analogues

The previous experiments revealed some substrate promiscuity for the A and C domain of FrsA, and the variety of FR derivatives isolated from A. crenata and C. vaccinii also indicate the acceptance and incorporation of structurally different substrates in vivo.^11,36 One natural FR derivate with an altered side chain has been isolated: FR-2 carries an acetyl moiety instead of the propionyl residue of N-Pp-Hle.¹¹ As acetyl-CoA is a very common product of glycolysis and a widely used precursor for C2 building

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blocks in different biosynthesis pathways,¹⁵⁷ it is most likely easily accessible for FR biosynthesis as well. As the Cstarter domains of FrsA and FrsD are 92.4% identical, while FrsD is supposed to catalyse the assembly of N-acetyl-hydroxyleucine (N-Ac-Hle), this also suggests the possible use of an activated acetyl residue, i.e., acetyl-CoA as an alternative substrate for the C domain.

We, therefore, performed the in vitro side chain assembly and transfer assay as described above but with different CoA substrates. As a positive control, we used propionyl-CoA, which leads to the already observed FR (m/z 1002.54) production. We exchanged propionyl-CoA for acetyl-CoA in order to produce FR-2 (m/z 988.53). Additionally, we tested butyryl-CoA to determine if a longer acyl chain is also accepted to form a new, unnatural “FR-butyryl” (m/z 1016.55). As negative controls, heat-inactivated proteins were used.

The results of the assays are pictured in Figure 4.23. The positive control with propionyl-CoA led to the production of FR and confirmed the results in section 4.4.3. The in vitro production of FR-2 was also successful since the mass and retention time fitted to the isolated standard of FR-2 (5). This proves the promiscuity of the Cstarter domain and might explain the detected high amounts of FR-2 in C. vaccinii cultures, as acetyl-CoA is easily accessible under laboratory conditions and hence can compete with propionyl-CoA. Interestingly, also the assay with butyryl-CoA yielded a novel signal for the calculated m/z of 1016.55. Its retention time was slightly higher than that of FR and in line with the earlier FR-2, fitting to the longer acyl chain. In Figure 4.24 the MS/MS fragmentation patterns of the novel compound and FR are depicted and compared. From m/z: 799.42 (loss of side chain, and loss of H2O, see Reher et al.),³⁶ the compounds have the same fragmentation pattern, indicating, that the additional methylation of FR (M+14 Da) is in the side chain and thus stems most likely from the incorporation of a butyryl group from the precursor butyryl-CoA in position (1) instead of the propionyl residue in FR like reported for FR-3 in position (2).³⁶

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Figure 4.23: Different Acyl-CoAs for in vitro side chain assembly and transfer assays with FrsA/B and FrsH. Extracted ion chromatograms of FR (m/z 1002.54), FR-2 (m/z 988.53) and FR-butyryl (m/z 1016.50)from HPLC-MS experiments; 1. FR standard (10 µg/ml); 2. FR-2 standard (10 µg/ml); 3. Purified FrsA/B, FrsH incubated with propionyl-CoA, L-Leu and FR-Core, and 4. negative control with heat-inactivated protein; 5. Purified FrsA/B, FrsH incubated with acetyl-CoA, L-Leu and FR-Core, and 6. negative control; 7. Purified FrsA/B, FrsH incubated with butyryl-CoA, L-Leu and FR-Core, and 8. negative control.

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Figure 4.24:MS/MS spectra of FR (m/z 1002.54) and FR-butyryl (m/z 1016.55). Fragmentation is labeled following the nomenclatur system by Ngoka et al. based on Biemanns modifications of Roepstorffs nomenclature in one-letter amino acid code.¹⁵⁵ b° = b-ion with loss of water. L’ = N-acetylhydroxyleucine, A = alanine, A’ = N-methylalanine, T’ = N,O-dimethylthreonine, T = threonine, L’’ = hydroxyleucine, L = leucine, F’ = phenyllactic acid, A’’ = N-methyldehydroalanine, L’’’ = N-propyonylhydroxyleucine, L^ = N-butyrylhydroxyleucine.

These data indicated the in vitro synthesis of a new FR derivative with an N-butyryl-3-hydroxyleucine side chain, but the in vitro assay yielded insufficient amounts of the compound for structure elucidation.

Upscaling of the in vitro assay was hampered by the low conversion rate of FR-Core to FR-butyryl and the limited supply of FR-Core. Thus, the next approach to gain access to FR-butyryl was the precursor-directed biosynthesis which is presented and discussed in 4.5.

We also tested if the substrate promiscuity of the A domain may lead to the integration of structurally different amino acids in the side chain assembly. For this assay, L-leucine, propionyl-CoA and FR-Core were used as a positive control to obtain FR and heat-inactivated proteins were used for the negative controls. The tested amino acids D-leucine and L-isoleucine are stereoisomers of L-leucine, so the resulting mass of the products would be expected to be the same as for FR, only the retention times might vary. As shown in Figure 4.25, the positive control generated FR and all negative controls showed no FR as expected. For the D-leucine, no signal for FR production was detected. This could imply, that the TE domain might act as a gatekeeper, a mechanism reported for a noncanonical TE domain in nocardicin biosynthesis, which only catalysed offloading of the NRPS when the β-lactamisation took place before.¹⁵⁸ An analogous phenomenon was reported for the TE domain in the biosynthesis of the glycopeptide antibiotic teicoplanin, where the TE domain is selective for cross-linked aglycones with

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an unusual extended N-terminal linker region.¹⁵⁹ In general, most TE domains are known so exert low substrate selectivity for the loading stage, giving way for pathway evolution, which is required to access new chemical diversity and maintain long term evolutionary fitness.⁴⁰ In the case of FrsATE, there is some substrate promiscuity visible, but the stereochemistry of the assembled side chain might be crucial for the activity of the TE domain. In contrast to the D-leucine, the assay with L-isoleucine resulted in a peak with the expected mass, so the steric organisation of the lipophilic side chain of the amino acid does not seem to be as influential as the first stereocenter for the activity of the TE domain. Interestingly, FrsH, which catalyses the hydroxylation at position 3 of leucine, also seems to be able to hydroxylate a tertiary carbon atom at position 3. Of course, these findings would need to be verified by more detailed structure elucidation of the product. However, similar to the FR-butyryl, this compound could not be isolated from the in vitro assay in sufficient amounts.

Figure 4.25: Different amino acids for in vitro side chain assembly and transfer assays with FrsA/B and FrsH. Extracted ion chromatograms of FR (m/z 1002.54) from HPLC-MS experiments; 1. FR standard (10 µg/ml); 2. Purified FrsA/B, FrsH incubated with propionyl-CoA, L-Leu and FR-Core, and 3. negative control with heat-inactivated protein; 4. Purified FrsA/B, FrsH incubated with propionyl-CoA, D-Leu and FR-Core, and 5. negative control; 6. Purified FrsA/B, FrsH incubated with propionyl-CoA, L-Ile and FR-Core, and 7. negative control.

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We also tested the substrate specificity of FrsATE concerning the acceptor substrate. Our previous results strongly suggested FR-Core to be the natural substrate of FrsATE, implying this step takes place after the cyclisation catalysed by FrsGTE. The linear depsipeptide product of FrsD-G might however also be a possible substrate, but as this substrate could not be obtained in the time of this thesis, we were not able to test it in the assay. Instead, we tested the minimal substrate 20 (L-Hle) to see if the TE domain might recognize this substrate and transfer the side chain onto the hydroxy group of Hle. The side chain harbours an additional free hydroxy group, so this reaction could take place repeatedly, resulting in oligomers. We analysed the LC-MS data from this assay for all possible masses, but no formation of any product was observed (see Figure 4.26 and Supplemental Figure 9.24).

Figure 4.26: Reaction scheme of the hypothetical intermolecular transesterification of 22 onto L-Hle (20).

It was also tested whether another cyclic depsipeptide containing a Hle moiety could serve as a substrate for FrsATE. The antibiotic natural product lysobactin (also known as katanosin B, 24) is commercially available and has a Hle moiety in its backbone. We performed the transesterification assay with lysobactin as substrate instead of FR-Core. The proposed reaction is shown in Figure 4.27. In this experiment also no formation of a new product could be observed (see Supplemental Figure 9.25), which is however not surprising, as the three-dimensional structure of lysobactin is vastly different to the one of FR-Core.

Figure 4.27: Reaction scheme of the hypothetical intermolecular transesterification of 22 onto lysobactin (24).

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The next step for this project would be the generation or isolation of the linear depsipeptide analogue to FR-Core and to use it as substrate for the assay, either as an open molecule, as SNAC or bound to the FrsG T domain. If the peptide is synthesized as CoA-thioester, the phosphopantetheinyl transferase Sfp of Bacillus subtillis could be used to load the substrate onto the expressed T domain,¹⁵⁰ an established method for TE domain analyses that was also used for NocBTE studies.¹⁵⁸ The T domain bound peptide would be the substrate closest to nature and could be also used to verify the function of FrsGTE. Another way to get hands on this linear molecule would be the cultivation of a frsGTE knock-out mutant. Without FrsGTE the biosynthesis should stop at the stage of the linear heptapeptide which might be released from the assembly line as such by other mechanisms like TE-type II mediated hydrolysis.¹⁶⁰ Otherwise, the side chain transesterification would take place independent from the cyclisation and yield a branched, linear FR molecule, which would disprove the hypothesis of FR-Core to be the natural substrate.

Additionally, a systematic evaluation of other, structurally diverse cyclic or linear peptide substrates is expected to shed more light on FrsATE acceptor specificity.

So far, our results support the hypothesis of the transesterifying TE domain and revealed some promiscuity for the composition of the side chain. This opens up new possibilities for biosynthetic engineering as these noncanonical intermolecular transesterification reactions can create depsipeptide bonds at polyketide or peptide side chain hydroxy functions. This biosynthetic principle can lead to the generation of potent natural products like salinamide A, which is produced by Sln9TE-catalysed transesterification in a marine Streptomyces bacterium and has strong antibiotic properties.⁴³ Besides Sln9TE and FrsATE, whose functions are now well investigated, there is another recently identified TE domain in the PKS/NRPS hybrid biosynthesis cluster of the cytotoxic necroximes that might have a similar function.¹⁶¹ Investigations of the nec BGC also revealed two TE domains. NecHTE is supposed to catalyse the cyclisation of the oxime-substituted benzolactone enamide core molecule Necroxime C and D and the monomodular NRPS NecA assembles a peptidic side chain, which is presumably transferred by NecATE to yield Necroxime A or B. A ΔnecA mutant abolished the production of Necroxime A and B which supports the theory of the NecA function, but the TE domain was not investigated in detail.¹⁶¹ Detailed comparative structural analyses of these three noncanonical TE domains could be a great basis for further exploitation efforts of this new type of enzyme for chemoenzymatic purposes. Our structural investigations of FrsATE are still ongoing and discussed in section 4.8.