© 2004 Oxford University Press
Short Communication |
His-404 and His-405 are Essential for Enzyme Catalytic Activities of a Bacterial Indole-3-Acetyl-L-Aspartic Acid Hydrolase
1 Department of Life Science, National Dong Hwa University, Hualien, Taiwan 97401, R.O.C.
2 Department of Biochemistry, University of Nevada, Reno, NV 89557, U.S.A.
3 Department of Horticultural Science, University of Minnesota, St. Paul, MN 55108, U.S.A.
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 Vmax (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.
Footnotes
4 Corresponding author: E-mail, jcchou{at}mail.ndhu.edu.tw; Fax, 886-3-863-3630.