His-404 and His-405 are essential for enzyme catalytic activities of a bacterial indole-3-acetyl-L-aspartic acid hydrolase
His-404 and His-405 are essential for enzyme catalytic activities of a bacterial indole-3-acetyl-L-aspartic acid hydrolase
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DOI：
10.1093/pcp/pch153
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发表时间：
2004-09-01
影响因子：
4.9
通讯作者：
Cohen, JD
中科院分区：
文献类型：
--
作者：
Chou, JC;Welch, WH;Cohen, JD
细菌吲哚-3-乙酰基-L-天冬氨酸（IAA-Asp）水解酶与拟南芥中发现的类似的IAA-氨基酸水解酶相比显示出非常高的底物特异性。相对于拟南芥衍生的酶，IAA-Asp水解酶还表现出非常高的V-max（快速反应速率）和更高的K-m（更低的底物亲和力）。这两个特征表明，该细菌酶和拟南芥酶之间的催化活性存在根本差异。利用计算机模拟的方法，发现假单胞菌非序列相关的锌依赖性外肽酶的催化残基His-385与IAA-Asp水解酶的His-405在结构上相匹配。His-405残基在细菌和拟南芥的所有相关序列中是保守的。该His-405对7种不同氨基酸的点突变实验导致酶活性的完全消除。然而，在邻近的His-404上的点突变到其他八个残基导致酶活性不同程度地降低。His-404的氨基酸取代也表明该残基影响IAA-Asp水解酶对底物IAA-Gly、IAA-Ala、IAA-Ser、IAA-Glu和IAA-Asn的较小活性。这些结果显示了结构建模的价值和潜力，预测目标残基的进一步研究和指导生物工程的酶的结构和功能。
Bacterial indole-3-acetyl-L-aspartic acid (IAA-Asp) hydrolase has shown very high substrate specificity compared with similar IAA-amino acid hydrolase enzymes found in Arabidopsis thaliana. The IAA-Asp hydrolase also exhibits, relative to the Arabidopsis thaliana-derived enzymes, a very high V-max (fast reaction rate) and a higher K-m (lower substrate affinity). These two characteristics indicate that there are fundamental differences in the catalytic activity between this bacterial enzyme and the Arabidopsis enzymes. By employing a computer simulation approach, a catalytic residue, His-385, from a non-sequence-related zinc-dependent exopeptidase of Pseudomonas was found to structurally match His-405 of IAA-Asp hydrolase. The His-405 residue is conserved in all related sequences of bacteria and Arabidopsis. Point mutation experiments of this His-405 to seven different amino acids resulted in complete elimination of enzyme activity. However, point mutation on the neighboring His-404 to eight other residues resulted in reduction, to various degrees, of enzyme activity. Amino acid substitutions for His-404 also showed that this residue influenced the minor activity of the IAA-Asp hydrolase for the substrates IAA-Gly, IAA-Ala, IAA-Ser, IAA-Glu and IAA-Asn. These results show the value and potential of structural modeling for predicting target residues for further study and for directing bioengineering of enzyme structure and function.