Candida albicans glucosamine-6-phosphate (GlcN-6-P) synthase was purified t
o apparent homogeneity with 52% yield from recombinant yeast YRSC-65 cells
efficiently overexpressing the GFA1 gene. The pure enzyme exhibited K-m(Gln
) = 1.56 mM and Km(Fru-6-P) = 1.41 mM and catalyzed GlcN-6-P formation with
k(cat) = 1150 min(-1). The isoelectric point of 4.6 +/- 0.05 was estimated
from isoelectric chromatofocusing, Gel filtration, native polyacrylamide g
el electrophoresis, subunit cross-linking, and SDS-polyacrylamide gel elect
rophoresis showed that the native enzyme was a homotetramer of 79.5-kDa sub
units, with an apparent molecular mass of 330-340 kDa, Results of chemical
modification of the enzyme by group-specific reagents established an essent
ial role of a cysteinyl residue at the glutamine-binding site and histidyl,
lysyl, arginyl, and tyrosyl moieties at the Flu-6-P-binding site. GlcN-6-P
synthase in crude extract was effectively inhibited by UDP-GlcNAc (IC50 =
0.67 mM). Purification of the enzyme markedly decreased the sensitivity to
the inhibitor, but this could be restored by addition of another effector,
glucose g-phosphate. Binding of UDP-GlcNAc to the pure enzyme in the presen
ce of Glc-6-P showed strong negative cooperativity, with RN = 0.54, whereas
in the absence of this sugar phosphate no cooperative effect was observed.
Pure enzyme was a substrate for cAMP-dependent protein kinase, the action
of which led to the substantial increase of GlcN-6-P synthase activity, cor
related with an extent of protein phosphorylation, The maximal level of act
ivity was observed for the enzyme molecules containing 1.21 +/- 0.08 mol of
phosphate/mol of GlcN-6-P synthase, Monitoring of GlcN-6-P synthase activi
ty and its sensitivity to UDP-GlcNAc during yeast --> mycelia transformatio
n of C, albicans cells, under in situ conditions, revealed a marked increas
e of the former and a substantial fall of the latter.