Critical aspartic acid residues in pseudouridine syntheses
Critical aspartic acid residues in pseudouridine syntheses
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DOI：
10.1074/jbc.274.32.22225
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发表时间：
1999-08-06
影响因子：
4.8
通讯作者：
Mueller, EG
中科院分区：
文献类型：
--
作者：
Ramamurthy, V;Swann, SL;Mueller, EG
伪尿嘧啶合成酶在某些RNA分子的特定位置催化尿嘧啶异构化为伪尿嘧啶。通过基因组数据库搜索和序列比对，Koonin （Koonin， E. V, (1996) Nucleic Acids Res. 24, 2411-2415）和Santi及其同事（Gustafsson， C,, Reid， R,, Greene， P. J, and Santi， D. V, (1996) Nucleic Acids Res. 24, 3756-3762）将这类酶分为四个家族，它们之间没有统计学上显著的全局序列相似性，经过进一步审查(Huang， H. L,；Pookanjanatavip， M., Gu， X， G., and Santi， D， V, (1998) Biochemistry 37, 344-351)， Santi小组发现一个天冬氨酸残基是所有排列序列中唯一存在的氨基酸；然后，他们证明了这种天冬氨酸残基在一个假尿嘧啶合成酶中具有催化作用。鉴于假尿嘧啶合成酶家族之间的全球差异，为了测试序列比对的功能意义，我们将另外两个家族的天冬氨酸残基分别改为丙氨酸和半胱氨酸：突变酶催化失活，但保留了结合tRNA底物的能力。我们还证实，突变酶从底物中释放尿嘧啶的速度与野生型假尿嘧啶合成酶的转化率相比并不显著。我们的结果清楚地表明，排列的天冬氨酸残基对这些酶的另外两个家族的假尿嘧啶合成酶的催化活性至关重要。支持序列比对的预测能力，并提示包含对齐的天冬氨酸残基的序列基序可能是假尿嘧啶合酶功能的先决条件。
The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V, (1996) Nucleic Acids Res. 24, 2411-2415) and, independently, Santi and co workers (Gustafsson, C,, Reid, R,, Greene, P. J., and Santi, D. V, (1996) Nucleic Acids Res. 24, 3756-3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other, Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X, G., and Santi, D, V, (1998) Biochemistry 37, 344-351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase, To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases, Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function.