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.

The Crystal and Solution Studies of Glucosamine-6-phosphate Synthase from Candida albicans

“…22,25 Crystallisation was attempted for the complete Gfa1p as well as its two domains: ISOM and GAH. Only ISOM gave crystals suitable for X-ray crystallographic measurement.…”

“…UDP-GlcNAc has been shown to inhibit glutamine hydrolysis and GlcN-6-P formation by the whole enzyme but has no effect on glutamine hydrolysis by the isolated GAH domain and on hexose phosphate isomerisation by the isolated ISOM domain. 22 Therefore, it is likely that the molecular base of the inhibitory effect of UDP-GlcNAc can be clearly seen only for the intact enzyme. The UDP-GlcNAc binding site is more than 10 Å from the isomerase active site.…”

O-GlcNAc signaling: a metabolic link between diabetes and cancer?

“…GFAT1 has two cyclic AMP dependent protein kinase (PKA) sites at serines 205 and 235. Whereas phosphorylation of S205 decreases cellular enzymatic activity, S235 phosphorylation does not affect activity20, 21. GFAT2 has a PKA site at serine 202 homologous to S205 of GFAT1, but PKA phosphorylation of this site increases its cellular activity22.…”

Engineering Candida albicans glucosamine‐6‐phosphate synthase for efficient enzyme purification

“…; Mouilleron et al ., ; Olchowy et al . ). Apparently, these consequences are more severe in the bacterial enzyme than in its fungal counterpart.…”

“…However, our results indicate that the consequences of an analogous modification of C. albicans GlcN-6-P synthase are somewhat different, as the specific activity of Gfa1-His 6 343p was only approximately 55% lower than that of WT Gfa1p, whereas in the case of its bacterial counterpart, the reduction in activity was approximately 99% (Richez et al 2007). Both effects might be explained as consequences of a possible disturbance of substrate channeling and interdomain communication that are crucial for the synthetic activity of GlcN-6-P synthase (Teplyakov et al 2001;Mouilleron et al, 2011;Olchowy et al 2007). Apparently, these consequences are more severe in the bacterial enzyme than in its fungal counterpart.…”