The gene for a specific IAA-asp hydrolase from Enterobacter agglomerans, iaaspH, is a potentially useful tool for modification of IAA homeostasis in higher plants that use the IAA-asp oxidation pathway for auxin catabolism. In order to optimize the utility of this gene for plant modification and to increase the success of obtaining iaaspH transformed plants from culture, we have investigated aspects of IAA-asp hydrolase catalysis. The catalytic characteristics of the IAA-asp hydrolase from Enterobacter agglomerans was studied using ten compounds that are structural analogues of IAA-asp. These compounds were tested as potential IAA-asp hydrolase substrates as well as for inhibition of IAA-asp hydrolysis. Among them, N-carbobenzyloxy-D-aspartic acid (N-CBZ-D-asp) and N-CBZ-L-asp were found to be the strongest inhibitors with more than 80% inhibition of IAA-asp hydrolysis. Aspartyl-L-aspartic acid and a asp-ser-asp-pro-arg peptide also showed strong inhibitory activities, reducing rates of IAA-L-asp hydrolysis, when added at equal molar amounts relative to the substrate, by 60% and 65%, respectively. N-CBZ-D-asp was chosen for further kinetic studies and for studies of its toxicity in relation to seed germination because it was a strong inhibitor, exhibited a very low rate of hydrolysis by the IAA-asp hydrolase and was commercially available. N-CBZ-D-asp was shown to be a competitive inhibitor for the Enterobacter agglomerans IAA-asp hydrolase with a Ki value of 1.22 mM. Studies of tomato seed germination showed that N-CBZ-D-asp did not affect the rate of seed germination at up to 1 mM, but the growth rate of seedlings was significantly reduced when the concentration in the medium was 0.5 mM and higher. These results indicate that, at suitable concentrations, N-CBZ-D-asp should be a useful tool for control of low level expression of the iaaspH in transgenic plants during critical stages of plant regeneration from culture.
Bibliographical noteFunding Information:
This work was supported by grant DE-FG02-00ER15079 from the U.S. Department of Energy and in part by the Minnesota Agricultural Experiment Station and the Gordon and Margaret Bailey Endowment for Environmental Horticulture.
- Auxin conjugates
- Enterobacter hydrolase
- Enzyme inhibitor
- Indole-3-acetylaspartic acid