Novel posttranslational activation of the LYS2-encoded alpha-aminoadipate reductase for biosynthesis of lysine and site-directed mutational analysis of conserved amino acid residues in the activation domain of Candida albicans

The alpha -aminoadipate pathway for lysine biosynthesis is present only in fungi. The alpha -aminoadipate reductase (AAR) of this pathway catalyzes th e conversion of ot-aminoadipic acid to alpha -aminoadipic-delta -semialdehy de by a complex mechanism involving two gene products, Lys2p and Lys5p. The LYS2 and LYS5 genes encode, respectively, a 155-kDa inactive AAR and a 30- kDa phosphopantetheinyl transferase (PPTase) which transfers a phosphopante theinyl group from coenzyme A (CoA) to Lys2p for the activation of Lys2p an d AAR activity. In the present investigation, we have confirmed the posttra nslational activation of the 150-kDa Lys2p of Candida albicans, a pathogeni c yeast, in the presence of CoA and C. albicans lys2 mutant (CLD2) extract as a source of PPTase (Lys5p). The recombinant Lys2p or CLD2 mutant extract exhibited no AAR activity with or without CoA. However, the recombinant 15 0-kDa Lys2p, when incubated with CLD2 extract and CoA, exhibited significan t AAR activity compared to that of wild-type C. albicans CAI4 extract. The PPTase in the CLD2 extract was required only for the activation of Lys2p an d not for AAR reaction. Site-directed mutational analysis of G882 and S884 of the Lys2p activation domain (LGGHSI) revealed no AAR activity, indicatin g that these two amino acids are essential for the activation. Replacement of other amino acid residues in the domain resulted in partial or full AAR activity. These results demonstrate the posttranslational activation and th e requirement of specific amino acid residues in the activation domain of t he AAR of C. albicans.