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Pyrrolysine
Pyrrolysine (symbol Pyl or O), encoded by the "amber" stop codon UAG, is a proteinogenic amino acid that is used in some methanogenic archaea and in bacteria. It consists of lysine with a 4-methylpyrroline-5-carboxylate in amide linkage with the εN of the lysine. Its pyrroline side-chain is similar to that of lysine in being basic and positively charged at neutral pH.
Nearly all genes are translated using only 20 standard amino acid building blocks. Two unusual genetically-encoded amino acids are selenocysteine and pyrrolysine. Pyrrolysine was discovered in 2002 at the active site of methyltransferase enzyme from a methane-producing archeon, Methanosarcina barkeri. This amino acid is encoded by UAG (normally a stop codon), and its synthesis and incorporation into protein is mediated via the biological machinery encoded by the pylTSBCD cluster of genes.
Pyrrolysine is synthesized in vivo by joining two molecules of L-lysine. One molecule of lysine is first converted to (3R)-3-methyl-D-ornithine, which is then ligated to a second lysine. An NH2 group is eliminated, followed by cyclization and dehydration step to yield L-pyrrolysine.
The extra pyrroline ring is incorporated into the active site of several methyltransferases, where it is believed to rotate relatively freely. It is believed that the ring is involved in positioning and displaying the methyl group of methylamine for attack by a corrinoid cofactor. The proposed model is that a nearby carboxylic acid bearing residue, glutamate, becomes protonated, and the proton can then be transferred to the imine ring nitrogen, exposing the adjacent ring carbon to nucleophilic addition by methylamine. The positively charged nitrogen created by this interaction may then interact with the deprotonated glutamate, causing a shift in ring orientation and exposing the methyl group derived from the methylamine to the binding cleft where it can interact with corrinoid. In this way a net CH+
3 is transferred to the cofactor's cobalt atom with a change of oxidation state from +1 to +3. The methylamine-derived ammonia is then released, restoring the original imine.
Unlike posttranslational modifications of lysine such as hydroxylysine, methyllysine, and hypusine, pyrrolysine is incorporated during translation (protein synthesis) as directed by the genetic code, just like the standard amino acids. It is encoded in mRNA by the UAG codon, which in most organisms is the 'amber' stop codon. This requires only the presence of the pylT gene, which encodes an unusual transfer RNA (tRNA) with a CUA anticodon, and the pylS gene, which encodes a class II aminoacyl-tRNA synthetase that charges the pylT-derived tRNA with pyrrolysine.
It was originally proposed that a specific downstream sequence "PYLIS", forming a stem-loop in the mRNA, forced the incorporation of pyrrolysine instead of terminating translation in methanogenic archaea. This would be analogous to the SECIS element for selenocysteine incorporation. However, the PYLIS model has lost favor in view of the lack of structural homology between PYLIS elements and the lack of UAG stops in those species.
The tRNA-aaRS pair for pyrrolysine ("orthogonal pair") is independent of other synthetases and tRNAs in most organisms including Escherichia coli, and further possesses some flexibility in the range of amino acids processed (the aaRS accepts some different molecules that are structurally similar to pyrrolysine), making it an attractive tool to allow the placement of a possibly wide range of functional chemical groups at arbitrarily specified locations in modified proteins. For example, the system provided one of two fluorophores incorporated site-specifically within calmodulin to allow the real-time examination of changes within the protein by FRET spectroscopy, and site-specific introduction of a photocaged lysine derivative. (See Expanded genetic code)
The recognition of a tRNA by an aaRS is by its acceptor stem sequence. The pyrrolysine tRNA can be modified to have an acceptor stem of another tRNA, allowing a different aaRS to act on it. In 2024, it was reported a version modified to accept alanine can effectively suppress premature termination codons (all three of them) in human cell lines.
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Pyrrolysine
Pyrrolysine (symbol Pyl or O), encoded by the "amber" stop codon UAG, is a proteinogenic amino acid that is used in some methanogenic archaea and in bacteria. It consists of lysine with a 4-methylpyrroline-5-carboxylate in amide linkage with the εN of the lysine. Its pyrroline side-chain is similar to that of lysine in being basic and positively charged at neutral pH.
Nearly all genes are translated using only 20 standard amino acid building blocks. Two unusual genetically-encoded amino acids are selenocysteine and pyrrolysine. Pyrrolysine was discovered in 2002 at the active site of methyltransferase enzyme from a methane-producing archeon, Methanosarcina barkeri. This amino acid is encoded by UAG (normally a stop codon), and its synthesis and incorporation into protein is mediated via the biological machinery encoded by the pylTSBCD cluster of genes.
Pyrrolysine is synthesized in vivo by joining two molecules of L-lysine. One molecule of lysine is first converted to (3R)-3-methyl-D-ornithine, which is then ligated to a second lysine. An NH2 group is eliminated, followed by cyclization and dehydration step to yield L-pyrrolysine.
The extra pyrroline ring is incorporated into the active site of several methyltransferases, where it is believed to rotate relatively freely. It is believed that the ring is involved in positioning and displaying the methyl group of methylamine for attack by a corrinoid cofactor. The proposed model is that a nearby carboxylic acid bearing residue, glutamate, becomes protonated, and the proton can then be transferred to the imine ring nitrogen, exposing the adjacent ring carbon to nucleophilic addition by methylamine. The positively charged nitrogen created by this interaction may then interact with the deprotonated glutamate, causing a shift in ring orientation and exposing the methyl group derived from the methylamine to the binding cleft where it can interact with corrinoid. In this way a net CH+
3 is transferred to the cofactor's cobalt atom with a change of oxidation state from +1 to +3. The methylamine-derived ammonia is then released, restoring the original imine.
Unlike posttranslational modifications of lysine such as hydroxylysine, methyllysine, and hypusine, pyrrolysine is incorporated during translation (protein synthesis) as directed by the genetic code, just like the standard amino acids. It is encoded in mRNA by the UAG codon, which in most organisms is the 'amber' stop codon. This requires only the presence of the pylT gene, which encodes an unusual transfer RNA (tRNA) with a CUA anticodon, and the pylS gene, which encodes a class II aminoacyl-tRNA synthetase that charges the pylT-derived tRNA with pyrrolysine.
It was originally proposed that a specific downstream sequence "PYLIS", forming a stem-loop in the mRNA, forced the incorporation of pyrrolysine instead of terminating translation in methanogenic archaea. This would be analogous to the SECIS element for selenocysteine incorporation. However, the PYLIS model has lost favor in view of the lack of structural homology between PYLIS elements and the lack of UAG stops in those species.
The tRNA-aaRS pair for pyrrolysine ("orthogonal pair") is independent of other synthetases and tRNAs in most organisms including Escherichia coli, and further possesses some flexibility in the range of amino acids processed (the aaRS accepts some different molecules that are structurally similar to pyrrolysine), making it an attractive tool to allow the placement of a possibly wide range of functional chemical groups at arbitrarily specified locations in modified proteins. For example, the system provided one of two fluorophores incorporated site-specifically within calmodulin to allow the real-time examination of changes within the protein by FRET spectroscopy, and site-specific introduction of a photocaged lysine derivative. (See Expanded genetic code)
The recognition of a tRNA by an aaRS is by its acceptor stem sequence. The pyrrolysine tRNA can be modified to have an acceptor stem of another tRNA, allowing a different aaRS to act on it. In 2024, it was reported a version modified to accept alanine can effectively suppress premature termination codons (all three of them) in human cell lines.