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In Methanosarcina acetivorans, the pyl operon consists of the pylS gene, which encodes for a class II aminoacyl-tRNA synthetase gene, pylT encoding a tRNA with a CUA anticodon and three other genes pylB, pylC, and pylD. PylS is able to directly aminoacylate the pylT gene product with pyrrolysine. PylS is only capable of attaching pyrrolysine to the tRNA and no other aminoacyl-tRNA synthetases can substitute in this process.In this study, a low molecular weight fraction from M. acetivorans was found to contain a substrate, presumbably pyrrolysine that PylS aminoacylated on to tRNAPyl. Similarly, a PylS substrate was seen in extracts of E. coli bearing pylBCD genes. This was seen using both the aminoacylation assay followed by Northerns of acid urea gel electrophoresis and the pyrophosphate exchange reaction. There was no detectable PylS substrate found in extracts from other organisms such as yeast, Bacillus, Salmonella, mice, and pigs. However, there was a detectable PylS substrate in extracts from the rumen wall of sheep but not the rumen contents of cattle.Examination of synthetic pyrrolysine showed that it could be reacted with ninhydrin, o-phthaldialdehyde (OPA), and sodium borohydride, sodium borodeuteride, and tritiated sodium borohydride. With these derivatized pyrrolysine products, pyrrolysine can be detected by thin layer chromatography and by high performance liquid chromatography. Mass spectral analysis of synthetic pyrrolysine established a fragmentation pattern with which to compare isolated PylS substrates, including the one seen in E. coli bearing pylBCD.To identify the nature of the PylS substrate from E. coli bearing copies of pylBCD, it needed to be purified away from inhibitory compounds that prevented E. coli growth when cellular extracts were added. Using a flash chromatography system based on a silica matrix and an elution gradient of ethyl acetate to methanol, partial purification was performed. Using this enriched fraction that was then added back to E. coli expressing pylT and pylS as well as mtmB, full-length MtmB was isolated. MtmB was subjected to mass spectral analysis and pyrrolysine was confirmed to be incorporated into the corresponding in-frame TAG in mtmB. This demonstrated that pyrrolysine was synthesized without a requirement for aminoacylation onto tRNAPyl or incorporation into protein and further modification. This was the first demonstration that complete pyrrolysine synthesis occurs with only pylBCD in E. coli. |
