Dissecting the roles of a strictly conserved tyrosine in substrate recognition and catalysis by pseudouridine 55 synthase.
Dissecting the roles of a strictly conserved tyrosine in substrate recognition and catalysis by pseudouridine 55 synthase.
复制标题
剖析严格保守的酪氨酸在假尿苷 55 合酶的底物识别和催化中的作用。
DOI：
10.1021/bi050961w
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发表时间：
2005
期刊：
影响因子：
2.9
通讯作者：
Huang,RavenH
中科院分区：
文献类型：
--
作者：
Phannachet,Kulwadee;Elias,Youssef;Huang,RavenH
Sequence alignment of the TruA, TruB, RsuA, and RluA families of pseudouridine synthases (ΨS) identifies a strictly conserved aspartic acid, which has been shown to be the critical nucleophile for the ΨS-catalyzed formation of pseudouridine (Ψ). 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 ofThermotoga maritimaandEscherichia colipseudouridine 55 synthase (Ψ55S) mutants in which the conserved Y is mutated to other amino acids. A new crystal structure of theT. maritimaΨ55S Y67F mutant in complex with a 5FU-RNA at 2.4 Å resolution revealed formation of 5-fluoro-6-hydroxypseudouridine (5FhΨ), the same product previously seen in wild-type Ψ55S−5FU-RNA complex structures. HPLC analysis confirmed efficient formation of 5FhΨ by both Ψ55S 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 ΨS 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 ΨS-catalyzed formation of Ψ.
