Date of this Version
Kalb, Madison. 2020. Characterization of Ralstonia solanacearum Isocyanide Hydratase Using Kinetic Analysis and Structure Determination. Undergraduate Honors Thesis. University of Nebraska-Lincoln.
Isocyanide hydratases (ICH) are unique enzymes within the DJ-1 superfamily that provide protection against toxic isocyanides by converting them to less toxic N-formamides. DJ-1 proteins are heavily researched as they have been shown to be involved in both cancer and Parkinson’s disease. These proteins crystallize and diffract under X-rays to high resolution, making them well-studied by protein crystallography, yet ICHs are an under-researched clade within this family. Previous research on ICH from Pseudomonas fluorescens (PfICH) revealed gated conformational dynamics of a mobile helix during catalysis, as well as provided insight into catalytically important residues.
This research aims to determine the structural and catalytic similarities and differences of PfICH and ICH from Ralstonia solanacearum (RsICH). Like PfICH, RsICH appears to also possess a mobile helix, although the relevance of this helix mobility for catalysis has not been tested. Kinetic analysis of RsICH showed that this enzyme is significantly faster than PfICH yet has much lower apparent affinity for the model substrate para-nitrophenyl isocyanide. Site directed mutagenesis of RsICH revealed the importance of residue E122 in catalysis, though the exact mechanism by which the glutamate provides the exhibited faster rates of reaction is still being investigated. Determination of the X-ray crystal structure of RsICH at 0.74 Å resolution revealed several residues with strong planarity deviations, as well as a well-conserved disulfide bond. Further research with RsICH will explore the relationship between conformational strain present in the resting enzyme and in catalysis, as well as the impact of post-translational modification of cysteines in RsICH conformation.