Journal Article

His-404 and His-405 are Essential for Enzyme Catalytic Activities of a Bacterial Indole-3-Acetyl-<span class="smallCaps">l</span>-Aspartic Acid Hydrolase

Jyh-Ching Chou, William H. Welch and Jerry D. Cohen

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 45, issue 9, pages 1335-1341
Published in print September 2004 | ISSN: 0032-0781
Published online September 2004 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pch153
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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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.

Keywords: Keywords: Auxin metabolism — Carboxypeptidase — Enzyme activity — Hydrolase — IAA conjugate.; Abbreviations: IAA-Asp, indole-3-acetyl-l-aspartic acid; IAASPH, indole-3-acetyl-l-aspartic acid hydrolase; IPTG, isopropyl-β-d-thiogalactopyranoside; TLC, thin-layer chromatography.

Journal Article.  3096 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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