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To establish that the chlorination detected on the 11.3 kDa KtzC domain was introduced on the tethered piperazyl group (rather than on some amino acid side chain of the thiolation domain), we subjected the products to action of the thioesterase enzyme TycF from the tyrocidin synthetase cluster.() This enzyme hydrolytically removes aminoacyl and peptidyl moieties from the phosphopantetheinyl arms of T domains as part of the normal chain release mechanism of nonribosomal peptide synthetases and is a mild (pH 7, room temperature) and selective route for deacylation. The low-molecular mass material thereby released was subjected to Fmoc derivatization on N1 of the piperazates to introduce a chromophore and facilitate detection by LC–MS. An N1-Fmoc-piperazate standard served as reference for release of unhalogenated piperazyl-S-KtzC which gave the anticipated peak by LC–MS analysis (Figure A). Most notably, upon TycF release of the (3S)-piperazyl-KtzC thioester from the halogenase incubations, a new peak in LC was detected (Figure A, trace ii), and high-resolution mass spectrometry showed this to be an Fmoc-chloropiperazate (observed mass of m/z 387.1141, expected mass of m/z 387.1112). In-depth analysis of the LC–MS data failed to show evidence of any hydroxylated products, confirming that KthP is highly selective for chlorination over hydroxylation. These TycF/Fmoc derivatization assays could also be used to show the anticipated requirement for O2 and for α-KG, previously established for this iron-dependent halogenase family (Figure S3 of the ).,, Consistent with the MALDI results, no chlorination product was detected from the substrate with the 3R configuration (trace iii).
the Outcome in Padanamide Biosynthesis
We then turned to 1H NMR spectroscopic analysis of the thioesterase-released chloropiperazate to help prove the regiochemistry of chlorination and relative stereochemistry. We began our NMR spectroscopic studies with analysis of the synthetic sample of Fmoc-protected (3S,5R)-5-chloropiperazate prepared according to the route shown in the . As is often the case for Fmoc-containing compounds, 1H NMR spectroscopic line shapes were extremely broad, precluding stereochemical analysis. In contrast, the TFA salt of (3S,5R)-5-chloropiperazic acid showed much better NMR spectroscopic properties and was thus chosen for analysis via double-quantum-filtered correlated spectroscopy (dqfCOSY). In small molecule structural analysis, dqfCOSY spectra exhibit a good signal-to-noise ratio down to nanomole amounts of analyte and allow determination of J coupling constants even for samples containing large amounts of impurities.() Analysis of the dqfCOSY spectrum of synthetic (3S,5R)-5-chloropiperazate (Figure S5 of the ) yielded J coupling constants for the protons at C3–C6 that unambiguously confirmed the 3S,5R stereochemistry of the synthetic sample and demonstrated the feasibility of using dqfCOSY spectra for determining the relative stereochemistry of chlorinated piperazic acid derivatives (Table S1 of the ).
Through their action, halogenases can convert simple amino acids into unique building blocks; in the model system presented by the kutzneride cluster, we see a broad display of Nature’s halogenation strategies. The kutzneride biosynthetic pathway is now shown, remarkably, to have four biosynthetic halogenases interspersed with the NRPS modules and the enzymes for providing the dedicated nonproteinogenic amino acid building blocks. All four of the halogenases use O2 as a cosubstrate, highlighting the oxidative nature of the four chlorinations. The enzymes are differentiated by the use of flavin cofactors (KtzQ and KtzR)() or nonheme iron cofactors (KtzD() and KthP) to tune the reactivity of the enzymes to match the electronic environments of their particular substrates. Halogenation can serve not only as an end in itself but also as an intermediate step in further diversification. In this case, chlorination by KthP tailors the piperazate scaffold with unique functionality, in addition to serving as a cue for subsequent stereochemical diversification of the residue. These results thus expand our knowledge of the important role halogenases play in the generation of the highly modified nonproteinogenic amino acids such as those found in the kutzneride antifungal scaffolds.
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To that end, - and -piperazic acids (3S and 3R, respectively) were converted to the corresponding piperazyl-CoAs as detailed in the and utilized as substrates for Sfp-mediated post-translational modification of the three different T domains. KtzC gave the best yield for the final product and was thus utilized in the following experiments (Figure S2 of the ). As shown in trace i of Figure B, MALDI MS analysis of the KtzC protein showed a mass of 10868.5 Da for the apo form of KtzC. When the Sfp-mediated post-translational modification used CoASH as a cosubstrate, the resulting holo HS-pantetheinyl form was detected (trace ii) with a mass of 11208.6 Da, showing the 342 Da (340 Da shift expected) mass shift from installation of the HS-pantetheinyl-P prosthetic group. When (3S)-piperazyl-CoA was used as a cosubstrate, the apo T domain was converted to a mass of 11320.8 Da (trace iii). The mass difference from the HS holo form was 112.2 Da, consistent with the expected 112 Da shift from the piperazyl group. Similarly, the (3R)-piperazyl-S-KtzC protein was generated from comparable incubations with (3R)-piperazyl-CoA (data not shown).
Patent US8962329 - Gene cluster - Google Patents
To test for piperazate halogenation activity of pure KthP, we conducted initial studies with free - or -piperazate. No conversion to chlorinated products could be detected by LC–MS analysis, following conversion of the cyclic hydrazo acids to their N1-Fmoc derivatives to generate a readily detected chromophore (Figure S2 of the ). The lack of activity on free piperazates was consistent with our prior studies on six mononuclear iron amino acyl halogenases, which demonstrated that they acted only on aminoacyl moieties tethered via a thioester linkage to the pantetheinyl arms of 8–10 kDa thiolation domains.()