BIOCHEMICAL CHARACTERIZATION OF AN EUCARIOTIC POLYKETIDE SYNTHASE

MARTIN SABATINI, SANTIAGO COMBA, SILVIA GRACIELA ALTABE, ANA ARABOLAZA, HUGO GRAMAJO

Congreso; SAIB 2017; 2017

Methyl-branched fatty acids (FA) are compounds which have numerousindustrial applications. They show improved physicochemicalproperties in comparison to linear carbon chain FA, the mostcommon FA found in natural sources. However, this kind of moleculesis extremely rare in nature. Thus, with the aim to develop newcompounds with optimized properties for industrial uses, in this workwe explored a new strategy to generate structural diversity in FA.Based on the mycoserosic acid synthase (MAS), a polyketidesynthase (PKS) from Mycobacterium tuberculosis that synthesizeslong chain methyl-branched fatty acids, we searched for a new PKScapable of using methylmalonyl-CoA (MMC) as extender unit. UsingMAS as query for a BLASTp search, we identified a new family ofun-characterized and highly conserved PKS in birds. The uropygialgland, in birds, is involved in the secretion of methyl-branched fattyacids, alcohols and esters that are used for cleaning and impermeabilizationof the plumage. We hypothesized that these compoundswould be produced by this new family of PKS. In particular, we startedthe characterization of a PKS enzyme, that we named ApMAS,from the duck Anas platyrhynchos; in this specie the uropygial secretionhas a high proportion of methyl-branched- C6 FA.PKS are enzymatic complexes that condense simple short chainacyl-CoA into larger molecules with diverse biological activities. Inan initial biochemical analysis of ApMAS, we determined the abilityof this enzyme to covalently bind to the substrates by using radiolabeledprecursors, acetyl-CoA/propionyl-CoA as starter units andmalonyl-CoA/MMC as extender units. Using this strategy we couldalso evidence the substrate transfer from an acyl-CoA to the ApMASacyl carrier protein domain (ACP). We then measured the kineticsof this transfer reaction catalized by the ApMAS acyltransferase domain.Finally, we could demonstrate the in vitro condensing ability ofthe ApMAS ketosynthase domain