Functional domains and interdomain communication in Candida albicans glucosamine-6-phosphate synthase
Résumé
Functional and structural properties of several truncated or mutated variants of Candida albicans glucosamine-6-phosphate synthase (Gfa1p) were compared to those of the wild-type enzyme. Fragments encompassing residues 1-345 and 346-712 of Gfa1p, expressed heterogenously in bacterial host as oligoHis fusions, were identified as functional domains: glutamine amide-hydrolysing (GAH) and hexose phosphate-isomerising (ISOM), respectively. It was found that native GAH is monomeric, while native ISOM forms tetramers, as does the whole enzyme. Spectrofluorimetric and kinetic studies of the isolated domains, the Δ218-283Gfa1p mutein and the whole enzyme revealed that the binding site for the feedback inhibitor, UDP-GlcNAc, is located in the ISOM domain. Inhibitor binding affects amidohydrolysing activity of GAH and, as a consequence, the GlcN-6-P-synthetic activity of the whole enzyme. The 218-283 fragment is neither involved in ligand binding nor in protein oligomerisation. Comparison of the catalytic activities of V711FGfa1p, Δ709-712Gfa1p, W97FGfa1p and W97GGfa1p to those of the native Gfa1p and the isolated domains provided evidence for an intramolecular channel connecting the GAH and ISOM domains of Gfa1p. The channel becomes leaky upon deletion of amino acids 709-712 and in W97F and W97G mutants. The W97 residue was found to function as a molecular gate, opening and closing the channel. The W97G and V711F substitutions resulted in an almost complete elimination of the GlcN-6-P-synthetic activity with the retention of the amidohydrolase and sugar phosphate-isomerising activities.
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