His-404 and His-405 are essential for enzyme catalytic activities of a bacterial indole-3-acetyl-L-aspartic acid hydrolase

被引:8
作者
Chou, JC [1 ]
Welch, WH
Cohen, JD
机构
[1] Natl Dong Hwa Univ, Dept Life Sci, Eugene, OR 97401 USA
[2] Univ Nevada, Dept Biochem, Reno, NV 89557 USA
[3] Univ Minnesota, Dept Hort Sci, St Paul, MN 55108 USA
关键词
auxin metabolism; carboxypeptidase; enzyme activity; hydrolase; IAA conjugate;
D O I
10.1093/pcp/pch153
中图分类号
Q94 [植物学];
学科分类号
071001 ;
摘要
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.
引用
收藏
页码:1335 / 1341
页数:7
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