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Literature summary for 6.1.1.26 extracted from
- Pyrrolysyl-tRNA synthetase an ordinary enzyme but an outstanding genetic code expansion tool (2014), Biochim. Biophys. Acta, 1844, 1059-1070 .
Application
| Application | Comment | Organism |
|---|---|---|
| molecular biology | PylRS as aminoacyl-tRNA synthetase allows the Pyl incorporation machinery to be easily engineered for the genetic incorporation of more than 100 non-canonical amino acids (NCAAs) or alpha-hydroxy acids into proteins at amber codon and the reassignment of other codons such as ochre UAA, opal UGA, and four-base AGGA codons to code NCAAs | Methanosarcina barkeri |
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene pylS, encoded in the pyl gene cluster | Methanosarcina mazei |
| gene pylS, encoded in the pyl gene cluster, DNA and amino acid sequence analysis, recombinant expression in Escherichia coli, the pyl gene cluster codes all necessary enzymes for the biosynthesis of Pyl from Escherichia coli metabolites | Methanosarcina barkeri |
Crystallization (Commentary)
| Crystallization (Comment) | Organism |
|---|---|
| apo-PylRS and PylRS complexes with different ligands, X-ray diffraction structure determination and analysis | Methanosarcina mazei |
| efforts to crystalize full-length PylRS are not successful due to the low solubility of the protein. The N-terminal domain is highly insoluble and aggregates full-length PylRS. But using the truncated C-terminal catalytic core of PylRS from Methanosarcina mazei, apo-PylRS and PylRS complexes with different ligands are successfully determined | Methanosarcina barkeri |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| additional information | engineering of mutants that display higher activities for their genetic incorporation than the wild-type PylRS and increased specificities, overview | Methanosarcina mazei |
| additional information | engineering of mutants that display higher activities for their genetic incorporation than the wild-type PylRS and increased specificities, overview | Methanosarcina barkeri |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | required | Methanosarcina mazei |  |
| Mg2+ | required | Methanosarcina barkeri | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + L-pyrrolysine + tRNAPyl | Methanosarcina mazei | - |
AMP + diphosphate + L-pyrrolysyl-tRNAPyl | - |
? | |
| ATP + L-pyrrolysine + tRNAPyl | Methanosarcina barkeri | - |
AMP + diphosphate + L-pyrrolysyl-tRNAPyl | - |
? | |
| ATP + L-pyrrolysine + tRNAPyl | Methanosarcina mazei ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88 | - |
AMP + diphosphate + L-pyrrolysyl-tRNAPyl | - |
? | |
| additional information | Methanosarcina mazei | unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate alpha-amine, and low selectivity toward the anticodon of tRNAPyl, overview. PylRS shows low selectivity toward the tRNA anticodon and low selectivity toward the Pyl alpha-amine | ? | - |
? | |
| additional information | Methanosarcina barkeri | unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate alpha-amine, and low selectivity toward the anticodon of tRNAPyl, overview. PylRS shows low selectivity toward the tRNA anticodon and low selectivity toward the Pyl alpha-amine | ? | - |
? | |
| additional information | Methanosarcina mazei ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88 | unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate alpha-amine, and low selectivity toward the anticodon of tRNAPyl, overview. PylRS shows low selectivity toward the tRNA anticodon and low selectivity toward the Pyl alpha-amine | ? | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Methanosarcina barkeri | Q6WRH6 | - |
- |
| Methanosarcina mazei | Q8PWY1 | - |
- |
| Methanosarcina mazei ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88 | Q8PWY1 | - |
- |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + L-pyrrolysine + tRNAPyl | - |
Methanosarcina mazei | AMP + diphosphate + L-pyrrolysyl-tRNAPyl | - |
? | |
| ATP + L-pyrrolysine + tRNAPyl | - |
Methanosarcina barkeri | AMP + diphosphate + L-pyrrolysyl-tRNAPyl | - |
? | |
| ATP + L-pyrrolysine + tRNAPyl | - |
Methanosarcina mazei ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88 | AMP + diphosphate + L-pyrrolysyl-tRNAPyl | - |
? | |
| additional information | unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate alpha-amine, and low selectivity toward the anticodon of tRNAPyl, overview. PylRS shows low selectivity toward the tRNA anticodon and low selectivity toward the Pyl alpha-amine | Methanosarcina mazei | ? | - |
? | |
| additional information | unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate alpha-amine, and low selectivity toward the anticodon of tRNAPyl, overview. PylRS shows low selectivity toward the tRNA anticodon and low selectivity toward the Pyl alpha-amine | Methanosarcina barkeri | ? | - |
? | |
| additional information | the recombinant pylS gene product charges tRNAPyl with Pyl, but the recombinant pylS gene product also succeeds in ligating [14C]Lys to tRNA. A mRNA secondary structure is definitely not necessary for the incorporation of Pyl at an amber codon. The binding of the side chain pyrroline of Pyl to the PylRS active site involves essentially van der Waals interactions. Replacing the side chain pyrroline with a similar size chemical component with a hydrophobic nature might retain the PylRS activity to aminoacylate tRNAPyl. PylRS weakly recognizes three non-canonical amino acids and mediates their incorporation into proteins at an amber codon in coordination with tRNAPyl, allowing the synthesis of proteins with site-specific lysine propionylation, butylation, and crotonylation | Methanosarcina barkeri | ? | - |
? | |
| additional information | the structure of Methanosarcina mazei PylRS catalytic core reveals a deep hydrophobic pocket for the binding of Pyl. Residues A302, L305, Y306, L309, N346, C348, and W417 form a bulky cavity for the binding of the side chain (4R,5R)-4-methyl-pyrroline-5-caboxylate of Pyl. The Pyl side chain also forms two hydrogen bonds at the PylRS active site, with one involving the side chain amide nitrogen of N346 and the Pyl side chain amide oxygen and the other involving the pyrroline nitrogen and the phenolic oxygen of Y384. Residue Y384 is at a flexible loop region that is random in the absence of Pyl but serves as a cap for the binding of Pyl to the active site. PylRS displays remarkably high tolerance toward variations of the substrate side chain, especially when a variation is at the pyrroline region. PylRS recognizes desmethyl-Pyl and is able to direct its incorporation at amber codon when in coordination with tRNAPyl. The binding of the side chain pyrroline of Pyl to the PylRS active site involves essentially van der Waals interactions. Replacing the side chain pyrroline with a similar size chemical component with a hydrophobic nature might retain the PylRS activity to aminoacylate tRNAPyl | Methanosarcina mazei | ? | - |
? | |
| additional information | unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate alpha-amine, and low selectivity toward the anticodon of tRNAPyl, overview. PylRS shows low selectivity toward the tRNA anticodon and low selectivity toward the Pyl alpha-amine | Methanosarcina mazei ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88 | ? | - |
? | |
| additional information | the structure of Methanosarcina mazei PylRS catalytic core reveals a deep hydrophobic pocket for the binding of Pyl. Residues A302, L305, Y306, L309, N346, C348, and W417 form a bulky cavity for the binding of the side chain (4R,5R)-4-methyl-pyrroline-5-caboxylate of Pyl. The Pyl side chain also forms two hydrogen bonds at the PylRS active site, with one involving the side chain amide nitrogen of N346 and the Pyl side chain amide oxygen and the other involving the pyrroline nitrogen and the phenolic oxygen of Y384. Residue Y384 is at a flexible loop region that is random in the absence of Pyl but serves as a cap for the binding of Pyl to the active site. PylRS displays remarkably high tolerance toward variations of the substrate side chain, especially when a variation is at the pyrroline region. PylRS recognizes desmethyl-Pyl and is able to direct its incorporation at amber codon when in coordination with tRNAPyl. The binding of the side chain pyrroline of Pyl to the PylRS active site involves essentially van der Waals interactions. Replacing the side chain pyrroline with a similar size chemical component with a hydrophobic nature might retain the PylRS activity to aminoacylate tRNAPyl | Methanosarcina mazei ATCC BAA-159 / DSM 3647 / Goe1 / Go1 / JCM 11833 / OCM 88 | ? | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| dimer | the PylRS catalytic core resembles that of other synthetases from the Class II AARS family. It has a typical beta-sheet core surrounded by several helices, forming a Rossmann fold for the binding of ATP. Like most other class II aminoacyl-tRNA synthetases, PylRS also forms an obligate dimer. Each subunit has an active site | Methanosarcina mazei |
| dimer | the PylRS catalytic core resembles that of other synthetases from the Class II AARS family. It has a typical beta-sheet core surrounded by several helices, forming a Rossmann fold for the binding of ATP. Like most other class II aminoacyl-tRNA synthetases, PylRS also forms an obligate dimer. Each subunit has an active site | Methanosarcina barkeri |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| PylRS | - |
Methanosarcina mazei |
| PylRS | - |
Methanosarcina barkeri |
| PylS | - |
Methanosarcina mazei |
| PylS | - |
Methanosarcina barkeri |
| pyrrolysyl-tRNA synthetase | - |
Methanosarcina mazei |
| pyrrolysyl-tRNA synthetase | - |
Methanosarcina barkeri |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| ATP | - |
Methanosarcina mazei | |
| ATP | - |
Methanosarcina barkeri | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| evolution | sequence alignment indicated that full-length PylRS contains a C-terminal class II AARS catalytic core and an N-terminal domain that apparently does not share sequence homology with any structurally known protein domains. The three dimensional organization of the PylRS catalytic core resembles that of other synthetases from the Class II AARS family | Methanosarcina mazei |
| evolution | sequence alignment indicated that full-length PylRS contains a C-terminal class II AARS catalytic core and an N-terminal domain that apparently does not share sequence homology with any structurally known protein domains. The three dimensional organization of the PylRS catalytic core resembles that of other synthetases from the Class II AARS family | Methanosarcina barkeri |
| additional information | along with its special CUA anticodon for the recognition of amber codon, the pylT transcript, tRNAPyl, has a distinct anticodon stem of six base pairs instead of five base pairs as observed in most tRNAs, a single base between D and anticodon stems, a single base between D and acceptor stems, and a three-base small variable arm | Methanosarcina mazei |
| additional information | along with its special CUA anticodon for the recognition of amber codon, the pylT transcript, tRNAPyl, has a distinct anticodon stem of six base pairs instead of five base pairs as observed in most tRNAs, a single base between D and anticodon stems, a single base between D and acceptor stems, and a three-base small variable arm | Methanosarcina barkeri |
| physiological function | the genetic incorporation of the 22nd proteinogenic amino acid, pyrolysine (Pyl) at amber codon is achieved by the action of pyrrolysyl-tRNA synthetase (PylRS) together with its cognate tRNAPyl | Methanosarcina mazei |
| physiological function | the genetic incorporation of the 22nd proteinogenic amino acid, pyrolysine (Pyl) at amber codon is achieved by the action of pyrrolysyl-tRNA synthetase (PylRS) together with its cognate tRNAPyl. When D-ornithine is provided in the growth medium, the clustered genes of pylT, pylS, pylC, and pylD were able to mediate amber suppression, pylC and pylD gene products are able to synthesize desmethyl-Pyl from D-ornithine and lysine and that PylRS tolerates alternative substrates | Methanosarcina barkeri |
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Release 2023.1 (January 2023)
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