The P450 bioI redox system.
thesisposted on 15.12.2014, 10:32 by Rachel Lawson
Cytochrome P450 BioI is isolated from the soil bacterium Bacillus subtilis. It is located in an operon that encodes genes involved in the synthesis of the vitamin biotin. From knock-out studies it was concluded that BioI catalyses an early step in the biosynthetic pathway, forming a precursor to the isolatable intermediate pimelic acid. This thesis covers the characterisation of this P450 and its various endogenous redox partners. The data presented here reports upon the bio-physical properties of the P450 including information on the redox potentials, how its environment affects its properties and the factors that govern substrate binding. To produce a catalytically active BioI redox system, two flavodoxins were cloned from B. subtilis and their over-expression and preparation is described. These flavodoxins have been characterised according to their redox and structural properties. The ability of the two flavodoxins to reconstitute the oxidase activity of BioI was examined by kinetic and spectroscopic techniques culminating in the analysis of the monooxygenation of various fatty acids. To complete the redox chain two NADPH-oxidoreductase candidates and a di-flavin reductase from B. subtilis were assessed and their ability to complement the P450 BioI-flavodoxin system examined.