165 Inhibition of isocitrate lyase shifts metabolism toward the isocitrate overproduction in yeast Yarrowia lipolytica

Abstract

ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20 and location. All but one (S846A) of these mutants had moderately to significantly reduced ATPase activities; how-ever, the PolyP distribution did not directly correlated to ATPase activity. As seen from Figure 1, membrane F796A mutant with the lowest activity had moderate increase in mid-chained PolyP2 (45–70 residues, attributed to nucleus) and PolyP3 (90–115, attributed to vacuole and secretory pathway), while L801A and especially E803A showed increase only for long-chained PolyP4–5 (≥170–200, attributed to cell envelope); these patters of PolyP4–5 were opposite to those in the C-tail mutants (Tomashevski & Petrov, 2015). Of the extracytosolic mutants, E847A with twofold reduced activity showed no changes in PolyP dis-tribution (Tomashevski & Petrov, 2013), while S846A with no change in ATPase activity exhibited 50–60% increase in short-chained PolyP1 (<25, attributed to cytosole and vacuole) and mid-chained PolyP3 and twofold drop of long-chained PolyP4–5. The S846A patterns were very similar to those of S911A. By contrast, T850A had moder-ate increase in PolyP1 and dramatic threefold jump in PolyP3; the ATPase activity decreased twofold. ATPase activity of D851A was very similar to T850A; nevertheless, changes in PolyP distribution were less prominent, although they were also found for mid-chained PolyP2 and 3. Changes in PolyP content in F796A could be explained by reduced ATPase activity and/or conformational changes (Petrov, 2010), which may lead to phosphate depositing in a form of PolyP. E803A was implicated in undercoupling between ATP hydrolysis and H + transport; thus twofold increase in PolyP4–5 may be explained not only in quan-titative but also in qualitative way. Results for replacement of the L9–10 loop residues seamed to be more tightly con-nected to their direct involvement in the enzyme phospho-rylation suggesting their stepwise and/or tandem involvement in this process. 0 50 100 150 200 250 300 F796A L801A E803A S846A T850A D851A PP1 PP2 PP3 PP4-5 ATPase Figure 1. Effect of the Pma1 point mutations on the PolyP distribution (% of wild type).