Abstract
Fatty acid-derived lipids are an important class of biomolecules. In addition to their primary role in cellular architecture, energy storage and signaling, they function in many other ways, e.g. as isolating or waterproof coatings, defense compounds and pheromones. Multiple enzymes mod- ify intermediates originating from fatty acid biosynthetic pathway, including desaturases (FADs) which synthesize unsaturated fatty acids, and reductases (FARs) which synthesize fatty alcohols.
Functionally highly diversified fatty acyl-modifying enzymes from insects, one of the most abun- dant animals in the world, present a significant source for modern biotechnology of cell factories.
This thesis summarizes available information on the FADs and FARs and describes the results which we have obtained while studying them.
In bumblebees, we identified several FAR transcripts which are abundantly expressed in male pheromone-producing tissue. We then functionally characterized the corresponding enzymes in yeasts, and estimated their participation in the biosynthesis of fatty alcohols observed in the mark- ing pheromone-producing tissue. The studied enzymes reduce broad range of substrates, from short fatty acyls (e.g. C14) to very long ones (e.g. C26), from saturated fatty acyls to polyunsatu- rated ones. We also found that expansion and subsequent diversification of male-specific bumblebee FARs might have been driven by transposable elements adjacent to the FAR genes.
In addition, we studied the effect of various residues in the substrate tunnel on the speci- ficity of the hornworm FADs. Previous studies have pinpointed that the specificity switch between evolutionarily related desaturases D2 and D3, which perform either Δ11/conjugase or Δ14reaction, respectively, is governed by a single residue at the bend of the substrate tunnel. We further ex- panded the set of residues of various properties at this position. Using both in vitrofunctional characterization in yeasts and molecular dynamics simulations, we found that smaller, more hy- drophilic residues, e.g. alanine and threonine, promote Δ11/conjugase reaction in the desaturase.
On the other hand, the bulkier, more hydrophobic residues, e.g. valine and isoleucine, promote Δ14 reaction.
We also focused on evolutionarily unrelated soluble desaturase from castor in which we dis- rupted the proton transfer chain (PTC). We exchanged polar residues in the PTC for their homolo- gous nonpolar counterparts from soluble methane monooxygenase (MMO). MMO shares common active site architecture with the desaturase, but performs hydroxylation rather than desaturation.
We found that the PTC mutations enhance minor hydroxylation channel in the desaturase, i.e. for- mation of alcohols, and they also promote formation of other minor oxygenated products, e.g. diols, allylic alcohols and epoxides.
