The second enzyme of histidine biosynthesis from Arabidopsis thaliana

Campbell, Samantha Allison (1999) The second enzyme of histidine biosynthesis from Arabidopsis thaliana. PhD thesis, University of Glasgow.

Abstract

The original objectives of the project were to clone the cDNAs for the first three enzymes of histidine biosynthesis from Arabidopsis and subsequently overexpress and characterise at least one of the enzymes. The cDNA for the second enzyme of the pathway, the bifunctional HisIE was the only cDNA for which viable clones were obtained. The cDNA for the Arabidopsis hisIE clone contained a chloroplastic targeting sequence which was removed in order to allow expression of the active domains of the HisIE protein. A construct which allowed overexpression of the HisIE protein to approximately 30% total cell protein was generated and the protein was subsequently purified to homogeneity using three chromatographic steps (ion exchange on DEAE, Phenyl Sepharose and gel filtration). The substrate PR-ATP was generated by biotransformation from ATP and PRPP using a purified HisG extract from E. coli and was purified by a new method using ion exchange chromatography and a volatile buffer system. This gave the first homogeneous sample of PR-ATP and allowed the kinetic characterisation of the enzyme. Optimal assay conditions for the Arabidopsis HisIE enzyme were established and basic kinetic parameters including the Km and kcat of the purified enzyme were determined. Chemical modification experiments with the histidine modifying reagent DEPC resulted in the rapid loss of enzyme activity even at very low concentrations. This suggested that a histidine residue might be involved in enzyme activity. To test this hypothesis site directed mutagenesis of the two conserved histidine residues within the protein was carried out and the mutant proteins overexpressed and purified. Subsequent characterisation of the mutant proteins and comparison of the Km and kcat with the wild type values indicated that neither of these histidine residues was catalytically important but suggested that one of the residues (H146) might be important for the quaternary structure of the HisIE enzyme and the other (H245) might sterically hinder the active site.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QH Natural history > QH345 Biochemistry
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: Coggins, Professor J.R.
• Date of Award: 1999
• Depositing User: Enlighten Team
• Unique ID: glathesis:1999-71586
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 10 May 2019 14:12
• Last Modified: 25 Oct 2022 15:14
• Thesis DOI: 10.5525/gla.thesis.71586
• URI: https://theses.gla.ac.uk/id/eprint/71586