title: His-404 and His-405 are essential for enzyme catalytic activities of a bacterial indole-3-acetyl-L-aspartic acid hydrolase.
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.
authors.affiliation: Department of Life Science, National Dong Hwa University, Hualien, Taiwan 97401, ROC. jcchou@mail.ndhu.edu.tw
authors.forename: Jyh-Ching
authors.initials: JC
authors.lastname: Chou
authors.forename: William H
authors.initials: WH
authors.lastname: Welch
authors.forename: Jerry D
authors.initials: JD
authors.lastname: Cohen
mesh_terms: D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D006639:Histidine / Q000378:metabolism*; D008969:Molecular Sequence Data; D017354:Point Mutation; D011549:Pseudomonas / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D006639:Histidine; D000619:Aminohydrolases