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Literature summary for 5.4.99.20 extracted from
- Molecular determinants for 23S rRNA recognition and modification by the E. coli pseudouridine synthase RluE (2018), J. Mol. Biol., 430, 1284-1294 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene rluE, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain AG1 (ME5305) | Escherichia coli |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| additional information | generation of the deletion mutants RluE DELTAN(1-35) lacking the N-terminal region deleted at residue 35 and RluE DELTAR158-K161 lacking loop L7-8. Mutant RluE DELTAN(1-35) shows pseudouridylation activity similar to wild-type, while the activity of mutant RluE DELTAR158-K161 is significantly reduced | Escherichia coli |
| R153A/P155G/R158A/R160A | site-directed mutagenesis, the mutant shows reduced activity compared to wild-type | Escherichia coli |
| R196A | site-directed mutagenesis, the mutant shows reduced activity compared to wild-type | Escherichia coli |
| R31A/R32A/K33A | site-directed mutagenesis, the mutant shows pseudouridylation activity similar to wild-type | Escherichia coli |
| R60A/K61A | site-directed mutagenesis, the mutant shows reduced activity compared to wild-type | Escherichia coli |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | dissociation constants (KD) of wild-type and mutant enzymes, kinetics, overview | Escherichia coli |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 23S rRNA uridine2457 | Escherichia coli | - |
23S rRNA pseudouridine2457 | - |
? |  |
| 23S rRNA uridine2457 | Escherichia coli K12 | - |
23S rRNA pseudouridine2457 | - |
? | |
| additional information | Escherichia coli | RluE acts as a stand-alone, highly specific enzyme forming the universally conserved pseudouridine at position 2457, located in helix 89 (H89) of the 23S rRNA in the peptidyltransferase center, detailed structure-function analysis to determine the structural elements both in RluE and in 23S rRNA required for RNA-protein interaction and pseudouridine formation | ? | - |
? | |
| additional information | Escherichia coli K12 | RluE acts as a stand-alone, highly specific enzyme forming the universally conserved pseudouridine at position 2457, located in helix 89 (H89) of the 23S rRNA in the peptidyltransferase center, detailed structure-function analysis to determine the structural elements both in RluE and in 23S rRNA required for RNA-protein interaction and pseudouridine formation | ? | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Escherichia coli | P75966 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain AG1 (ME5305) by nickel affinity chromatography, ultrafiltration, and gel filtration | Escherichia coli |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 23S rRNA uridine2457 | - |
Escherichia coli | 23S rRNA pseudouridine2457 | - |
? | |
| 23S rRNA uridine2457 | Escherichia coli pseudouridine synthase RluE targeting a single uridine in helix 89 (H89) in the peptidyltransferase center (PTC) of 23S rRNA. The target of RluE in H89 is Psi2457, analysis, overview | Escherichia coli | 23S rRNA pseudouridine2457 | - |
? | |
| 23S rRNA uridine2457 | - |
Escherichia coli K12 | 23S rRNA pseudouridine2457 | - |
? | |
| 23S rRNA uridine2457 | Escherichia coli pseudouridine synthase RluE targeting a single uridine in helix 89 (H89) in the peptidyltransferase center (PTC) of 23S rRNA. The target of RluE in H89 is Psi2457, analysis, overview | Escherichia coli K12 | 23S rRNA pseudouridine2457 | - |
? | |
| additional information | RluE acts as a stand-alone, highly specific enzyme forming the universally conserved pseudouridine at position 2457, located in helix 89 (H89) of the 23S rRNA in the peptidyltransferase center, detailed structure-function analysis to determine the structural elements both in RluE and in 23S rRNA required for RNA-protein interaction and pseudouridine formation | Escherichia coli | ? | - |
? | |
| additional information | RluE recognizes a large part of 23S rRNA comprising both H89 and the single-stranded flanking regions which explains the high substrate specificity of RluE. Within RluE, the target RNA is recognized through sequence-specific contacts with loop L7-8 as well as interactions with loop L1-2 and the flexible N-terminal region | Escherichia coli | ? | - |
? | |
| additional information | RluE acts as a stand-alone, highly specific enzyme forming the universally conserved pseudouridine at position 2457, located in helix 89 (H89) of the 23S rRNA in the peptidyltransferase center, detailed structure-function analysis to determine the structural elements both in RluE and in 23S rRNA required for RNA-protein interaction and pseudouridine formation | Escherichia coli K12 | ? | - |
? | |
| additional information | RluE recognizes a large part of 23S rRNA comprising both H89 and the single-stranded flanking regions which explains the high substrate specificity of RluE. Within RluE, the target RNA is recognized through sequence-specific contacts with loop L7-8 as well as interactions with loop L1-2 and the flexible N-terminal region | Escherichia coli K12 | ? | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| RluE | - |
Escherichia coli |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 37 | - |
assay at | Escherichia coli |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 1.7 | - |
23S rRNA uridine2457 | pH 7.5, 37°C | Escherichia coli |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7.5 | - |
assay at | Escherichia coli |
General Information
| General Information | Comment | Organism |
|---|---|---|
| evolution | pseudouridylation is catalyzed by two different systems: stand-alone pseudouridine synthases found in all domains of life and H/ACAribonucleoproteins (RNPs) that are only present in archaea and eukaryotes. All pseudouridine synthases are classified into six families based on the presence of common motifs, which are named after representative enzymes, namely, RluA, RsuA, TruA, TruB, TruD and Pus10 | Escherichia coli |
| physiological function | Pseudouridines are not only found in rRNA but in all types of cellular RNA including mRNAs, tRNAs, small nuclear RNAs, small nucleolar RNAs and long-noncoding RNAs. In general, pseudouridines are thought to play a role in RNA stabilization, structure and correct RNA folding. RluE is a faster pseudouridine synthase than other enzymes which likely enables it to act in the early stages of ribosome formation. Molecular mechanism of RluE forming a highly conserved pseudouridine during ribosome biogenesis, overview | Escherichia coli |
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