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PDBsum entry 2ab4
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Isomerase/RNA
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PDB id
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2ab4
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References listed in PDB file
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Key reference
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Title
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Dissecting the roles of a strictly conserved tyrosine in substrate recognition and catalysis by pseudouridine 55 synthase.
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Authors
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K.Phannachet,
Y.Elias,
R.H.Huang.
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Ref.
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Biochemistry, 2005,
44,
15488-15494.
[DOI no: ]
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PubMed id
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Abstract
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Sequence alignment of the TruA, TruB, RsuA, and RluA families of pseudouridine
synthases (PsiS) identifies a strictly conserved aspartic acid, which has been
shown to be the critical nucleophile for the PsiS-catalyzed formation of
pseudouridine (Psi). However, superposition of the representative structures
from these four families of enzymes identifies two additional amino acids, a
lysine or an arginine (K/R) and a tyrosine (Y), from a K/RxY motif that are
structurally conserved in the active site. We have created a series of
Thermotoga maritima and Escherichia coli pseudouridine 55 synthase (Psi55S)
mutants in which the conserved Y is mutated to other amino acids. A new crystal
structure of the T. maritima Psi55S Y67F mutant in complex with a 5FU-RNA at 2.4
A resolution revealed formation of 5-fluoro-6-hydroxypseudouridine (5FhPsi), the
same product previously seen in wild-type Psi55S-5FU-RNA complex structures.
HPLC analysis confirmed efficient formation of 5FhPsi by both Psi55S Y67F and
Y67L mutants but to a much lesser extent by the Y67A mutant when 5FU-RNA
substrate was used. However, both HPLC analysis and a tritium release assay
indicated that these mutants had no detectable enzymatic activity when the
natural RNA substrate was used. The combined structural and mutational studies
lead us to propose that the side chain of the conserved tyrosine in these four
families of PsiS plays a dual role within the active site, maintaining the
structural integrity of the active site through its hydrophobic phenyl ring and
acting as a general base through its OH group for the proton abstraction
required in the last step of PsiS-catalyzed formation of Psi.
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