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J Biol Chem, Vol. 274, Issue 7, 4000-4008, February 12, 1999

Oligomeric Structure and Regulation of Candida albicans Glucosamine-6-phosphate Synthase

Sawomir Milewski, Danuta Kuszczak, Robert Jdrzejczak, Rachel J. Smith^¶, Alistair J. P. Brown^¶, and Graham W. Gooday^¶

From the  Department of Pharmaceutical Technology and Biochemistry, Technical University of Gdask, 11/12 Narutowicza Street, 80-952 Gdask, Poland and the ^¶ Department of Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom

Candida albicans glucosamine-6-phosphate (GlcN-6-P) synthase was purified to 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 K_m(Fru-6-P) = 1.41 mM and catalyzed GlcN-6-P formation with k_cat = 1150 min¹. The isoelectric point of 4.6 ± 0.05 was estimated from isoelectric chromatofocusing. Gel filtration, native polyacrylamide gel electrophoresis, subunit cross-linking, and SDS-polyacrylamide gel electrophoresis showed that the native enzyme was a homotetramer of 79.5-kDa subunits, with an apparent molecular mass of 330-340 kDa. Results of chemical modification of the enzyme by group-specific reagents established an essential role of a cysteinyl residue at the glutamine-binding site and histidyl, lysyl, arginyl, and tyrosyl moieties at the Fru-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 6-phosphate. Binding of UDP-GlcNAc to the pure enzyme in the presence of Glc-6-P showed strong negative cooperativity, with n_H = 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, correlated with an extent of protein phosphorylation. The maximal level of activity 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 activity and its sensitivity to UDP-GlcNAc during yeast  mycelia transformation of C. albicans cells, under in situ conditions, revealed a marked increase of the former and a substantial fall of the latter.

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