Modification of quaternary structure of Candida albicans GlcN-6-P synthase and its desensitization to inhibition by UDP-GlcNAc by site-directed mutagenesis.

Site-directed mutagenesis of the CaGFA1 gene encoding glucosamine-6-phosphate synthase from Candida albicans was performed. Desensitization of the enzyme to inhibition by UDPGlcNAc was achieved upon T487I and H492F substitutions at the UDP-GlcNAc binding site, exchange of D524, S525 and S527 for Ala at the dimer:dimer interface and construction of the tail-lock array (L434R and L460A) at the C-tail region. The first two sets if mutageneses but not the last one resulted in conversion of the tetrameric enzyme into its dimeric form. Evidence for links and communication between the UDP-GlcNAc binding site and the dimer-dimer contact areas are presented. The CaGfa1-T487IH492F and CaGfa1-KHSH-D524AS525AS527A muteins are the first examples of the successful conversion of eukaryotic GlcN-6-P synthase into its prokaryotic-like version upon rational site-directed mutagenesis.