Structural and kinetics properties of a mutated phytoene desaturase from Rubrivivax gelatinosus with modified product specificity

The phytoene desaturase CrtI from Rubrivivax gelatinosus catalyzes simultaneously a three- and four-step desaturation producing both neurosporene and lycopene. These carotenes are intermediates for the synthesis of spheroidene and spirilloxanthin, respectively. Two different mutation libraries for the crtI gene from R. gelatinosus were constructed to screen for modified enzymes which synthesize almost exclusively either neurosporene or lycopene. The resulting mutants carried between one and four amino acid exchanges and at least one of them affected the secondary protein structure by shortening or extending one of the helices. A prominent amino acid which was exchanged in the neurosporene or lycopene-forming desaturase was leucine 208. Enzyme kinetic studies were carried out with the L208 modified desaturase and the specificities for phytoene and neurosporene as substrates determined. Higher and lower values correlate well with the higher or lower potential for the synthesis of lycopene from neurosporene. TopPred analysis of the mutations of L208 indicated that the location is in a highly hydrophobic membrane-integrated region which is a good candidate for the substrate-binding site of the desaturase.

Research highlights
► CrtI phytoene desaturase mutants exhibit amino acid exchanges in helix regions together with a modified product specificity.
► Enzyme kinetic properties were changed with respect either to neuropsorene or lycopene synthesis.
► A membrane spanning model of CrtI with functional sites is proposed.
► The mutants mimic the evolution of CrtI desaturases in purple bacteria.