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

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

Research output: Contribution to journalArticle

10 Scopus citations

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 languageEnglish (US)
Pages (from-to)1335-1341
Number of pages7
JournalPlant and Cell Physiology
Volume45
Issue number9
DOIs
StatePublished - Sep 2004

Keywords

  • Auxin metabolism
  • Carboxypeptidase
  • Enzyme activity
  • Hydrolase
  • IAA conjugate

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