Use this URL to cite or link to this record in EThOS:
Title: Over-expression and characterisation of Brassica napus and Escherichia coli 3-oxoacyl-[acyl carrier protein] reductase
Author: Thomas, Neil Ciaron
ISNI:       0000 0001 3524 6711
Awarding Body: Durham University
Current Institution: Durham University
Date of Award: 1999
Availability of Full Text:
Access from EThOS:
Access from Institution:
A full length cDNA clone of Brassica napus 3-oxoacyl-ACP reductase (β-ketoacyl-ACP reductase; E.C.; βKR) and the Escherichia coli gene for the same enzyme, have been over-expressed in E. coli. Both the Brassica napus seed and Escherichia coli βKR proteins have been purified by a rapid two-step, single chromatography matrix method. Glutaraldehyde cross-linking studies show the plant βKR is expressed as a tetramer and the E. coli enzyme is expressed as a dimer. The secondary structure of the two proteins was predicted via analysis of circular dichroism spectra, which also show dilution dependent unfolding of a-helical structure in the plant enzyme, a possible explanation for the dilution inactivation of βKRs. Ultrafiltration substrate binding studies and a bireactant initial velocity study show that Brassica napus βKR employs a fixed order ternary complex mechanism with NADPH binding to the enzyme first. One-dimensional western blot analysis indicates two isoforms of βKR (28 kDa and 31 kDa) in crude B. napus seed extracts. Further analysis using two- dimensional western blots demonstrates the presence of four major isoforms. Comparison with 2D blots from B. campestris suggests that one of the major isoforms has originated from that source. The crystal structure of the E. coli βKR enzyme is also discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Isoforms; Kinetic mechanism