A conserved aspartate of tRNA pseudouridine synthase is essential for activity and a probable nucleophilic catalyst
A conserved aspartate of tRNA pseudouridine synthase is essential for activity and a probable nucleophilic catalyst
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DOI：
10.1021/bi971874
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发表时间：
1998-01-06
期刊：
影响因子：
2.9
通讯作者：
Santi, DV
中科院分区：
文献类型：
--
作者：
Huang, LX;Pookanjanatavip, M;Santi, DV
tRNA假尿苷合酶I在许多tRNA的反密码子环中的35、39和/或40位催化尿苷转化为假尿苷。将假尿苷合酶I克隆在T7启动子后，并在大肠杆菌中表达至约20%的总可溶性蛋白质，氟尿嘧啶取代的tRNA引起假尿苷合酶I的时间依赖性失活，并与涉及位置39处的FUMP的酶形成共价复合物。Asp 60在所有已知和推定的假尿苷激酶中保守，突变为具有不同侧链的氨基酸。所有Asp 60突变体结合tRNA，但无催化活性，未能与氟尿嘧啶取代的tRNA形成共价复合物。我们的结论是保守的Asp 60是必不可少的假尿苷合酶的活性，并提出机制，涉及这个残基的重要催化作用。
tRNA pseudouridine synthase I catalyzes the conversion of uridine to pseudouridine at positions 35, 39, and/or 40 in the anticodon loop of many tRNAs. Pseudouridine synthase I was cloned behind a T7 promoter and expressed in Escherichia coli to about 20% of total soluble proteins, Fluorouracil-substituted tRNA caused a time-dependent inactivation of pseudouridine synthase I and formed a covalent complex with the enzyme that involved the FUMP at position 39. Asp60, conserved in all known and putative pseudouridine synthases, was mutated to amino acids with diverse side chains. All Asp60 mutants bound tRNA but were catalytically inactive and failed to form covalent complexes with fluorouracil-substituted tRNA. We conclude that the conserved Asp60 is essential for pseudouridine synthase activity and propose mechanisms which involve this residue in important catalytic roles.