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Information on EC 5.4.99.23 - 23S rRNA pseudouridine1911/1915/1917 synthase
for references in articles please use BRENDA:EC5.4.99.23
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EC Tree 5 Isomerases
5.4 Intramolecular transferases
5.4.99 Transferring other groups
5.4.99.23 23S rRNA pseudouridine1911/1915/1917 synthase
IUBMB Comments
Pseudouridine synthase RluD converts uridines at positions 1911, 1915, and 1917 of 23S rRNA to pseudouridines. These nucleotides are located in the functionally important helix-loop 69 of 23S rRNA .
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Reaction Schemes
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23S rRNA uridine1911/uridine1915/uridine1917
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23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
Synonyms
pseudouridine synthase rlud, rlud pseudouridine synthase, more
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
RluD
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
23S rRNA uridine1911/uridine1915/uridine1917 = 23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
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SYSTEMATIC NAME
IUBMB Comments
23S rRNA-uridine1911/1915/1917 uracil mutase
Pseudouridine synthase RluD converts uridines at positions 1911, 1915, and 1917 of 23S rRNA to pseudouridines. These nucleotides are located in the functionally important helix-loop 69 of 23S rRNA [1].
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
23S rRNA uridine1911/uridine1915/uridine1917
23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
additional information
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Substrates: the recombinant chimeric enzyme mutant RluCD isomerizes many uridines of rRNA in a non-specific manner
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23S rRNA uridine1911/uridine1915/uridine1917
23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
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Substrates: -
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23S rRNA uridine1911/uridine1915/uridine1917
23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
Substrates: -
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23S rRNA uridine1911/uridine1915/uridine1917
23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
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Substrates: -
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23S rRNA uridine1911/uridine1915/uridine1917
23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
Substrates: -
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23S rRNA uridine1911/uridine1915/uridine1917
23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
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Substrates: pseudouridine synthase RluD converts uridines at positions 1911, 1915, and 1917 of 23S rRNA to pseudouridines. These nucleotides are located in the functionally important helix-loop 69 of 23S rRNA. RluD is the only pseudouridine synthase that is required for normal growth in Escherichia coli
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23S rRNA uridine1911/uridine1915/uridine1917
23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
Substrates: RluD is far more efficient in modifying uridine residues in helix 69 in structured 50S subunits, compared to free or synthetic 23S rRNA. It is suggested that pseudouridine modifications in helix 69 are made late in the assembly of 23S rRNA into mature 50S subunits
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23S rRNA uridine1911/uridine1915/uridine1917
23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
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Substrates: pseudouridine synthase RluD converts uridines at positions 1911, 1915, and 1917 of 23S rRNA to pseudouridines. These nucleotides are located in the functionally important helix-loop 69 of 23S rRNA. RluD isomerizes uridine in vivo only at positions 1911, 1915, and 1917, regardless of the presence of uridine at other positions in the loop of helix 69 of 23S rRNA variants. Substitution of one uridine by cytosine has no effect on the conversion of others (i.e. formation of pseudouridines at positions 1911, 1915, and 1917 are independent of each other). The substrate specificity of the pseudouridine synthase RluD is analyzed in vivo by using 23S rRNA variants. A1916 is the only position in the loop of helix 69, where mutations affect the RluD specific pseudouridine formation
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23S rRNA uridine1911/uridine1915/uridine1917
23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
Substrates: RluD is far more efficient in modifying uridine residues in helix 69 in structured 50S subunits, compared to free or synthetic 23S rRNA
Products: -
Products: -
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23S rRNA uridine1911/uridine1915/uridine1917
23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
Substrates: the enzyme is responsible in vivo for synthesis of the three pseudouridines clustered in a stem-loop at positions 1911, 1915, and 1917 of 23S RNA
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23S rRNA uridine1911/uridine1915/uridine1917
23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
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Substrates: -
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
23S rRNA uridine1911/uridine1915/uridine1917
23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
23S rRNA uridine1911/uridine1915/uridine1917
23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
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Substrates: -
Products: -
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23S rRNA uridine1911/uridine1915/uridine1917
23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
Substrates: -
Products: -
Products: -
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23S rRNA uridine1911/uridine1915/uridine1917
23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
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Substrates: -
Products: -
Products: -
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23S rRNA uridine1911/uridine1915/uridine1917
23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
Substrates: -
Products: -
Products: -
?
23S rRNA uridine1911/uridine1915/uridine1917
23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
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Substrates: pseudouridine synthase RluD converts uridines at positions 1911, 1915, and 1917 of 23S rRNA to pseudouridines. These nucleotides are located in the functionally important helix-loop 69 of 23S rRNA. RluD is the only pseudouridine synthase that is required for normal growth in Escherichia coli
Products: -
Products: -
?
23S rRNA uridine1911/uridine1915/uridine1917
23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
Substrates: RluD is far more efficient in modifying uridine residues in helix 69 in structured 50S subunits, compared to free or synthetic 23S rRNA. It is suggested that pseudouridine modifications in helix 69 are made late in the assembly of 23S rRNA into mature 50S subunits
Products: -
Products: -
?
23S rRNA uridine1911/uridine1915/uridine1917
23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
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Substrates: -
Products: -
Products: -
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DISEASE
TITLE OF PUBLICATION
LINK TO PUBMED
Infections
Francisella tularensis live vaccine strain folate metabolism and pseudouridine synthase gene mutants modulate macrophage caspase-1 activation.
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Highest Expressing Human Cell Lines
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
the structure of RluD (a RluA family member) emphasizes that the RluA, RsuA, TruB, and TruA families of pseudouridine synthases arose by divergent evolution from a common ancestor
malfunction
physiological function
malfunction
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a mutation that suppresses the growth defect of an rluD mutant localizes to prfB, which encodes peptide chain release factor RF2
malfunction
a truncation mutant of the gene for RluD (responsible for synthesis of 23S rRNA pseudouridines 1911, 1915, and 1917) blocks pseudouridine formation and inhibits growth. RluD mutants D139T and D139N are completely inactive in vivo and in vitro. In vivo, the growth defect can be completely restored by transformation of an RluD-inactive strain with plasmids carrying genes for RluD D139T or RluD D139N. Pseudouridine sequencing of the 23S rRNA from these transformed strains demonstrates the lack of these pseudouridines. Pseudoreversion is not responsible because transformation with empty vector under identical conditions does not alter the growth rate
malfunction
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loss of the RluD protein and its product pseudouridine residues (pseudouridine1911, pseudouridine 1915 and pseudouridine 1917) in a deletion strain lacking the rluD gene. This strain exhibits defects in ribosome assembly, biogenesis, and function. Specifically, there is a deficit of 70S ribosomes, an increase in 50S and 30S subunits, and the appearance of new 62S (derived from the breakdown of unstable 70S ribosomes) and 39S particles (immature precursors of the 50S subunits). The defect in ribosome assembly and resulting growth phenotype of the mutant could be restored by expression of wild-type RluD and synthesis of C1911, C1915, and C1917 residues, but not by catalytically inactive mutant RluD proteins, incapable of pseudouridine formation
malfunction
the absence of RluD results in severe growth inhibition. Both the absence of pseudouridine and the growth defect can be reversed by insertion of a plasmid carrying the rluD gene into the mutant cell, clearly linking both effects to the absence of RluD. Growth inhibition may be due to the lack of one or more of the 23S RNA pseudouridines made by this synthase since pseudouridines 1915 and 1917 are universally conserved and are located in proximity to the decoding center of the ribosome where they could be involved in modulating codon recognition
malfunction
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deletion of the rluD gene in Salmonella enterica has negligible effects on growth, ribosomal subunit association, or stop codon readthrough
malfunction
excessive pseudouridylation of 23S rRNA by the chimeric mutant RluCD reduces progression of ribosome assembly during early or middle stages. Modification of sites in 23S rRNA prevents ribosome assembly, interfering positions are located inside the ribosome, mapping. It is plausible that pseudouridines can cause RNA misfolding when present at non-native positions. Recombinant expression of RluCD protein itself inhibits ribosome assembly by binding to the precursor particles and blocking the assembly of r-proteins. The phenotypic effects are caused by the catalytic activity of the chimeric pseudouridine synthase RluCD, mechanism, overview. The excessive pseudouridines in rRNA species cause strong selection against 70S ribosome pool. Ribosome assembly defect causes degradation of unassembled rRNA and accumulation of small RNA fragments on the top of gradient
physiological function
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at least one of the three pseudouridine bases appears to have a function in termination of translation
physiological function
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pseudouridine synthase RluD converts uridines at positions 1911, 1915, and 1917 of 23S rRNA to pseudouridines. These nucleotides are located in the functionally important helix-loop 69 of 23S rRNA. RluD is the only pseudouridine synthase that is required for normal growth in Escherichia coli. RluD directed isomerization of uridines occurs as a late step during the assembly of the (50S) large ribosomal subunit
physiological function
RluD is a ribosomal assembly factor that may be involved in the late stages of maturation of the large ribosomal subunit
Show AA Sequence and Transmembrane Helices (902 entries)
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PDB
SCOP
CATH
UNIPROT
ORGANISM
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
monomer
analytical ultracentrifugation sedimentation velocity experiments show that RluD is monomeric in solution
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
2.0 A structure of the catalytic domain of RluD (residues 77326). The catalytic domain folds into a mainly antiparallel beta-sheet flanked by several loops and helices. A positively charged cleft that presumably binds RNA leads to the conserved Asp139. The RluD N-terminal S4 domain, connected by a flexible linker, is disordered in our structure. RluD is very similar in both catalytic domain structure and active site arrangement to the pseudouridine synthases RsuA, TruB, and TruA
crystallization of selenomethionine-substituted RluD by the hanging-drop method, crystals diffract to 1.9 A and belong to space group P4(3)2(1)2, with unit cell parameters a = b = 75.14, c = 181.81 A
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crystals of full-length RluD are grown at 20°C using the hanging drop method. The S4 domain of RluD appears to be highly flexible or unfolded and is completely invisible in the electron density map
crystals of SeMet-labeled DELTARluD are obtained under oil by the microbatch method, crystal structure of the catalytic module of RluD (residues 68326, DELTARluD) refined at 1.8 A to a final R-factor of 21.8%. DELTARluD is a monomeric enzyme having an overall mixed alpha/beta fold. The DELTARluD molecule consists of two subdomains, a catalytic subdomain and C-terminal subdomain with the RNA-binding cleft formed by loops extending from the catalytic sub-domain. Comparison of the structure with other pseudouridine synthases
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D139N
D139T
mutation completely blocks pseudouridine formation in vivo and in vitro. The mutant rluD gene produces a protein capable of complete reversal of the growth defect (of Escherichia coli mutant with a truncation in the rluD gene) without concomitant pseudouridine formation
additional information
construction of a chimeric pseudouridine synthase (RluCD) containing the N-terminal S4 domain of enzyme RluC (EC 5.4.99.24) and the C-terminal catalytic domain of enzyme RluD (EC 5.4.99.23). The chimeric mutant is able to introduce excessive pseudouridines into rRNA at non-native positions. The chimeric enzyme RluCD is used as a tool to study an effect of over-modification of rRNA on the ribosome biogenesis. Excessive pseudouridylation of 23S rRNA reduces progression of ribosome assembly during early or middle stages. A modification interference approach identifies the sites in 23S rRNA whose modification prevents ribosome assembly. It is plausible that pseudouridines can cause RNA misfolding when present at non-native positions. RluCD isomerizes many uridines of rRNA in a non-specific manner. Induction of the RluCD at the exponential growth phase leads to severe inhibition of translation while transcription is only slightly affected
D139N
mutation completely blocks pseudouridine formation in vivo and in vitro. The mutant rluD gene produces a protein capable of complete reversal of the growth defect (of Escherichia coli mutant with a truncation in the rluD gene) without concomitant pseudouridine formation
D139N
site-directed mutagenesis of the chimeric enzyme mutant RluCD, the mutant is catalytically inactive
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant expression of chimeric enzyme mutants RluCD in Escherichia coli strains M15 of CD204, expression of chimeric pseudouridine synthase RluCD, under control of arabinose inducible araBAD promoter, interferes with ribosome formation in wild-type M15 strain and strain CD204 lacking RluC and RluD, while cells transformed with catalytically inactive RluCD (RluCD D139N) and empty vector (pQE60), used as a controls, represent a normal ribosomal profile in sucrose density gradient. Strain CD204 contains a mutant form of RF-2 (D131Y) that suppresses ribosome assembly defect caused by the deletion of the RluD. Strain rluD114 where yfiI (RluD) gene has been disrupted with Km cassette is used as a parent strain to construct strain CD204
wild-type and mutant enzymes D139T and D139N are overexpressed from plasmids
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Del Campo, M.; Ofengand, J.; Malhotra, A.
Purification and crystallization of Escherichia coli pseudouridine synthase RluD
Acta Crystallogr. Sect. D
59
1871-1873
2003
Escherichia coli
brenda
Sivaraman, J.; Iannuzzi, P.; Cygler, M.; Matte, A.
Crystal structure of the RluD pseudouridine synthase catalytic module, an enzyme that modifies 23S rRNA and is essential for normal cell growth of Escherichia coli
J. Mol. Biol.
335
87-101
2004
Escherichia coli (Q8X9F0), Escherichia coli
brenda
Gutgsell, N.S.; Deutscher, M.P.; Ofengand, J.
The pseudouridine synthase RluD is required for normal ribosome assembly and function in Escherichia coli
RNA
11
1141-1152
2005
Escherichia coli
brenda
Leppik, M.; Peil, L.; Kipper, K.; Liiv, A.; Remme, J.
Substrate specificity of the pseudouridine synthase RluD in Escherichia coli
FEBS J.
274
5759-5766
2007
Escherichia coli
brenda
Huang, L.; Ku, J.; Pookanjanatavip, M.; Gu, X.; Wang, D.; Greene, P.J.; Santi, D.V.
Identification of two Escherichia coli pseudouridine synthases that show multisite specificity for 23S RNA
Biochemistry
37
15951-15957
1998
Escherichia coli
brenda
Mizutani, K.; Machida, Y.; Unzai, S.; Park, S.Y.; Tame, J.R.
Crystal structures of the catalytic domains of pseudouridine synthases RluC and RluD from Escherichia coli
Biochemistry
43
4454-4463
2004
Escherichia coli (P33643)
brenda
Wrzesinski, J.; Bakin, A.; Ofengand, J.; Lane, B.G.
Isolation and properties of Escherichia coli 23S-RNA pseudouridine 1911, 1915, 1917 synthase (RluD)
IUBMB Life
50
33-37
2000
Escherichia coli (P33643)
brenda
Ejby, M.; Sorensen, M.A.; Pedersen, S.
Pseudouridylation of helix 69 of 23S rRNA is necessary for an effective translation termination
Proc. Natl. Acad. Sci. USA
104
19410-19415
2007
Escherichia coli
brenda
Del Campo, M.; Ofengand, J.; Malhotra, A.
Crystal structure of the catalytic domain of RluD, the only rRNA pseudouridine synthase required for normal growth of Escherichia coli
RNA
10
231-239
2004
Escherichia coli (P33643), Escherichia coli
brenda
Vaidyanathan, P.P.; Deutscher, M.P.; Malhotra, A.
RluD, a highly conserved pseudouridine synthase, modifies 50S subunits more specifically and efficiently than free 23S rRNA
RNA
13
1868-1876
2007
Escherichia coli (P33643), Escherichia coli
brenda
Raychaudhuri, S.; Conrad, J.; Hall, B.G.; Ofengand, J.
A pseudouridine synthase required for the formation of two universally conserved pseudouridines in ribosomal RNA is essential for normal growth of Escherichia coli
RNA
4
1407-1417
1998
Escherichia coli (P33643)
brenda
Gutgsell, N.S.; Del Campo, M.; Raychaudhuri, S.; Ofengand, J.
A second function for pseudouridine synthases: A point mutant of RluD unable to form pseudouridines 1911, 1915, and 1917 in Escherichia coli 23S ribosomal RNA restores normal growth to an RluD-minus strain
RNA
7
990-998
2001
Escherichia coli (P33643)
brenda
OConnor, M.; Gregory, S.T.
Inactivation of the RluD pseudouridine synthase has minimal effects on growth and ribosome function in wild-type Escherichia coli and Salmonella enterica
J. Bacteriol.
193
154-162
2011
Salmonella enterica
brenda
Leppik, M.; Liiv, A.; Remme, J.
Random pseuoduridylation in vivo reveals critical region of Escherichia coli 23S rRNA for ribosome assembly
Nucleic Acids Res.
45
6098-6108
2017
Escherichia coli (P33643)
brenda
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Release 2023.1 (January 2023)
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