REPURPOSING A NON-RIBOSOMAL PEPTIDE SYNTHETASE (TYROCIDINE SYNTHETASE A) FOR AMINOACYL COA BIOSYNTHESISByRuth Njeri MuchiriIn Taxus plants, the biosynthesis of the pharmaceutical paclitaxel includes the transfer of β-amino-β-phenylpropanoyls from coenzyme A to the diterpenoid baccatin III by an acyl CoA-dependent acyltransferase. The CoA ligase that biosynthesizes key β-amino-β-phenylpropanoyl CoAs in Taxus plants has not yet been isolated or characterized. In a screen for an alternative catalyst, a multienzyme, nonribosomal peptidyl synthetase on the pathway that produces tyrocidines was identified as a surrogate CoA ligase. The tridomain starter module (Phe–ATE) of the tyrocidine synthetase A normally activates (S)-α-phenylalanine to an adenylate phosphate anhydride in the adenylation domain. The activated phenylalanine intermediate is then thioesterified by the pendent (i.e. covalent) pantetheine attached to the adjacent thiolation domain. In the current project, the adenylation and thiolation domains were found to function as a CoA ligase, making α-, β-phenylalanyl, and, more importantly, phenylisoserinyl CoA. The latter two are known substrates of a phenylpropanoyltransferase (BAPT) on the biosynthetic pathway of the antimitotic paclitaxel. Tyc(Phe–AT) was used in additional specificity studies with a focus on arylisoserines, since the corresponding CoA thioesters are important for biosynthesizing precursors of the paclitaxel analogs (such as the prostate cancer drug carbazitaxel). The stereospecificity of Tyc(Phe–AT) for the various stereoisomers of phenylisoserine was explored, showing reduced turnover for the (2R,3R) isomer, and no turnover with (2S,3R)-phenylisoserine relative to the benchmark (2R,3S)-isomer. The latter (2R,3S)-diastereoisomer of phenylisoserine matches the stereochemistry of the natural side chain of paclitaxel. Thus, our preliminary work evaluated the substrate specificity of Tyc(Phe–AT) (taking advantage of its enantiospecificity) for racemates of (2S,3R)- and (2R,3S)-arylisoserine. Structure-activity relationship analyses in earlier, independent studies showed that arylisoserine is necessary for the effective anticancer activity of paclitaxel. To access these activated CoA intermediates biosynthetically for use in a coupled enzyme assay with the paclitaxel pathway-specific 13-O-phenylpropanoyltransferase (BAPT), the CoA ligase function of Tyc(Phe–AT) was employed to convert various aryl- and non-aryl isoserine analogs to their CoA thioesters. We propose the products of the Tyc(Phe–AT) reaction can be transferred to baccatin III by a permissive BAPT. The isoserine substrates were synthesized by the Staudinger reaction that formed the Schiff base between benzaldehyde analogs and p-anisidine. This base was then reacted with acetoxyacetyl chloride in the presence of triethylamine. The amino and hydroxyl groups of the lactam product of this reaction were deprotected. Finally, hydrolysis of the lactam produced the isoserine analog. Tyc(Phe–AT) catalysis converted these analogs to their corresponding isoserinyl CoAs. All substrates in which the phenyl ring was substituted at ortho- (F, Cl, NO2), para- (F, Cl, Br, Me, OH, and NO2), or meta- (F, Cl, Br, Me, OH, CH3O and NO2) were converted to their CoA thioesters. Activity was also observed with the non-aromatic β-(cyclohexyl)isoserine and heteroaromatic β-(thiophenyl)isoserine analogs, but not with the aliphatic alkanyl groups (isopropyl-, and tert-butyl isoserine). This work provides a stepping stone towards novel biosynthesis of paclitaxel analogs with better efficacy than the parent drug.
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