The Properties of the Fatty Aldehyde Decarbonylase from Synechocystis PCC6803
Kalibala, Robert James
Thesis or dissertation
University of Exeter
Reason for embargo
Publication arising from research
Alkanes dominate the constituents of gasoline, diesel, and jet fuel and are naturally produced by diverse species; saturated and unsaturated fatty acids are converted to alkanes and alkenes respectively by the enzyme aldehyde decarbonylase (AD). Here we describe the over-expression, purification, data collected and X-ray crystal structure solved for the AD protein from Synechocystis PCC6803. This report describes the optimisation of over-expression, protein purification and characterization and crystallisation of the Synechocystis cyanobacterial AD enzyme (SynADC) has been carried out. The optimisation of protein expression has been carried out using the pET160, pET22b and pColdTM II. Expression of soluble protein was obtained with all vectors. The initial LumioTM tag on pET160 prevented the protein from crystallising; the pColdTM II vector with a small His-tag was used for high soluble protein over-expression. The purification of the SynADC was optimized and the enzyme was characterised biochemically, SynADC was found to be a dimer of 29 kDa molecular weight. Metal contents were investigated using ICP-MS, SynADC protein was found to contain; Zn, Fe, Ni and Mn metals in a ratio (2.37, 1.16, 0.137, and 0.032) mg/l respectively. The enzyme has been assayed using a series of ferredoxin assays of (C8, C10, C12, C13, C16 and C18) and activity has been determined using C13 aldehyde and C18 aldehyde. The enzyme has been successfully crystallised with four different ligands (valeric acid, Hexanoic acid, C4 and C8) using the microbatch method and metal soaking, this has allowed the X-ray structure to be determined. Based on this structure predication of electron transfer mechanism, a mutagenesis experiment has been carried out with the change of Asp143 to Asn, Leu and Ala. The enzyme has been assayed using PMS. Experiments to determine potential proteins, which could interact with SynADC, have been carried out. Positive results have been obtained using SDS-PAGE however, more protein is required for mass spectrometric determination. This project was part of a larger study to clone and solve the structure of the Synechocystis Cyanobacterial AD in order to understand its substrate specificity and mechanism. Work carried out in collaboration with others is clearly mentioned in this thesis.
MbyRes in Biosciences