Functional domains and interdomain communication in Candida albicans glucosamine-6-phosphate synthase

Abstract

Functional and structural properties of several truncated or mutated variants of Candida albicans Gfa1p (glucosamine-6-phosphate synthase) were compared with those of the wild-type enzyme. Fragments encompassing residues 1-345 and 346-712 of Gfa1p, expressed heterogeneously in bacterial host as His6 fusions, were identified as the functional GAH (glutamine amidehydrolysing) and ISOM (hexose phosphate-isomerizing) domains respectively. It was found that the native GAH domain is monomeric, whereas the native ISOM domain forms tetramers, as does the whole enzyme. Spectrofluorimetric and kinetic studies of the isolated domains, the Delta218-283Gfa1p mutein and the wild-type enzyme revealed that the binding site for the feedback inhibitor, uridine 5'-diphospho-N-acetyl-D-glucosamine, is located in the ISOM domain. Inhibitor binding affects amidohydrolysing activity of the GAH domain and, as a consequence, the GlcN-6-P (D-glucosamine-6-phosphate)-synthetic activity of the whole enzyme. The fragment containing residues 218-283 is neither involved in ligand binding nor in protein oligomerization. Comparison of the catalytic activities of Gfa1p(V711F), Delta709-712Gfa1p, Gfa1p(W97F) and Gfa1p(W97G) with 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 the W97F and W97G mutants. The Trp97 residue was found to function as a molecular gate, opening and closing the channel. The W97G and V711F mutations resulted in an almost complete elimination of the GlcN-6-P-synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities.

Figure 1. Alignment of amino acid sequences of C. albicans and E. coli GlcN-6-P synthases

The linker between the GAH and ISOM domains of E. coli GlcN-6-P synthase and the residues substituted in the recombinant versions of the C. albicans enzyme, constructed for the purpose of the present study, are indicated. Numbering for the C. albicans Gfa1p is shown. White letters on a black background indicate conserved residues. Ca, C. albicans; Ec, E. coli.

Figure 2. Stereo view of the three dimensional structure of E. coli GlcN-6-P synthase

The functional domains, some relevant fragments and the intramolecular channel (small spheres) are indicated. The image has been generated from PDB ID 1JXA [5].

Figure 3. SDS/PAGE analysis of the purification of N-terminally His₆-tagged residues 346–712 of Gfa1p overexpressed in E. coli

Lane M, molecular-mass markers (in kDa); lane 1, total lysate of E. coli BL21(DE3)pLysS pET23b-FRU cells; lane 2, total lysate of control E. coli BL21(DE3)pLysS pET23b cells; lanes 3 and 4, wash fractions with buffer W; and, lane 5, fraction eluted by 0.5 M imidazole.

Figure 4. Changes in enzymatic activities of GAH-His₆, Gfa1p and S208AGfa1p upon phosphorylation with PKA

Samples of Gfa1p, S208AGfa1p or GAH-His₆ were incubated for 30 min in the presence of indicated compounds. A specific kinase A inhibitor, H-89 (10 μg/ml), was then added and the samples were assayed for amidohydrolysing and/or GlcN-6-P-synthetic activity.

Figure 5. SDS/PAGE analysis of GAHp–His₆, Gfa1p and Gfa1p^S208A phosphorylation by PKA

Mixtures obtained after a 30 min incubation of a given protein with indicated components were separated by SDS/PAGE. (A) Gel stained with the GelCode™ kit. (B) The same gel as (A) stained with Coomassie Brilliant Blue. Lane 1, GAHp–His₆ and PKA; lane 2, GAHp–His₆; lane 3, GAHp–His₆, PKA and H-89; lane 4, Gfa1p and PKA; lane 5, Gfa1p, PKA and 10 mM Fru-6-P; lane 6, S208AGfa1p and PKA; lane 7, Gfa1p. Each mixture contained 10 μM cAMP, 1 mM ATP, 10 mM EDTA and 40 mM NaF. Protein load: 10 μg (lanes 1–3) and 3 μg (lanes 4–7). M, molecular mass markers (from top to bottom): 120 kDa, 100 kDa, 85 kDa, 70 kDa, 60 kDa, 50 kDa and 40 kDa.

Figure 6. Time course of ammonia release from L-Gln, catalysed by Gfa1p and its mutants

Enzyme samples were incubated with 10 mM L-Gln and the released ammonia was quantified with the Nessler's reagent. The results are shown as means±S.D. for experiments performed in triplicate.

Figure 7. Blockage of the interdomain channel in Gfa1p upon V711F substitution

(A) Computed structure created by homology modelling using E. coli GlcN-6-P synthase (1JXA) [5] as a template. (B) Putative result of V711F substitution. Residues marked with an asterisk (*) are from the neighbouring subunit. The channel is indicated by black spheres.