Characterization of Aldehyde dehydrogenases A and B from Pseudomonas syringae DC3000 through Site-Directed Mutagenesis and X-Ray Crystallography

Kristy Witte

Abstract

This project examines the importance of aldehyde dehydrogenases A and B (AldA and AldB) in the bacterial biosynthesis of the plant growth hormone, auxin. Pseudomonas syringae, a bacterial plant pathogen that degrades the cell wall employs aldehyde dehydrogenases in the synthesis of auxin in the form of Indole-3-acetic acid (IAA) from Indole-3-acetaldehyde (IAAld). The aim of this project is to use site-directed mutagenesis to examine the role of various amino acids in the active sites of AldA and AldB. In addition to site-directed mutagenesis, DNA plasmid mini-prep, DNA gel electrophoresis, and DNA Polymerase Chain Reaction were carried out to isolate the mutated DNA product. Nanodrop was used to determine product concentration and purity ratios. DNA sequencing and SDS-PAGE gels were used to verify mutation before protein purification was performed using a nickel column. X-ray crystallography was then used to determine crystal structure and enzyme assays were performed to measure enzyme activity. DNA sequencing results showed the absence of the desired mutation for two of the samples and the presence of the mutation for one. Site-directed mutagenesis will be repeated to generate additional desired mutations to further examine.

Faculty Advisor: Soon Goo Lee

Acknowledgment: Dr. Soon Goo Lee, Marissa Vincent

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