Abstract
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 Km (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.
Original language | English (US) |
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Pages (from-to) | 1335-1341 |
Number of pages | 7 |
Journal | Plant and Cell Physiology |
Volume | 45 |
Issue number | 9 |
DOIs | |
State | Published - Sep 2004 |
Bibliographical note
Funding Information:This work was supported by U.S. DOE grant DE-FG02-00ER15079 to J.D.C. and by R.O.C. National Science Council grant NSC-91-2311-B-259-004 to J.C.C.
Keywords
- Auxin metabolism
- Carboxypeptidase
- Enzyme activity
- Hydrolase
- IAA conjugate