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Literature summary extracted from

  • Olchowy, J.; Gabriel, I.; Milewski, S.
    Functional domains and interdomain communication in Candida albicans glucosamine-6-phosphate synthase (2007), Biochem. J., 404, 121-130.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.6.1.16 expressed in Escherichia coli strain BL21(DE3) pLysS Candida albicans

Protein Variants

EC Number Protein Variants Comment Organism
2.6.1.16 additional information expression of truncated enzyme variants as His-tagged proteins. Fragments encompassing residues 1-345 and 346-712 represent the functional glutamine amide-hydrolysing GAH and hexose phosphate-isomerizing domains ISOM, respectively. The native GAH domain is monomeric, whereas the native ISOM domain forms tetramers, as does the whole enzyme. 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 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. An intramolecular channel connects the GAH and ISOM domains. The channel becomes leaky upon deletion of amino acids 709-712 and in the W97F and W97G mutants Candida albicans
2.6.1.16 V711F mutation results in an almost complete elimination of the GlcN-6-P-synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities Candida albicans
2.6.1.16 V711F reduction of the glucosamine 6-phosphate-synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities Candida albicans
2.6.1.16 W97F mutation results in an almost complete elimination of the GlcN-6-P synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities Candida albicans
2.6.1.16 W97F reduction of the glucosamine 6-phosphate-synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities. Residue W97 functions as a molecular gate, opening and closing the intramolecular channel that connects the glutamine amide-hydrolysing and hexose phosphate-isomerizing domains Candida albicans
2.6.1.16 W97G mutation results in an almost complete elimination of the GlcN-6-P-synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities Candida albicans
2.6.1.16 W97G almost complete elimination of the glucosamine 6-phosphate-synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities. Residue W97 functions as a molecular gate, opening and closing the intramolecular channel that connects the glutamine amide-hydrolysing and hexose phosphate-isomerizing domains Candida albicans

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.6.1.16 0.31
-
 L-gamma-glutamyl-p-nitroanilide residues 1-345, His-tagged glutamine amide-hydrolysing domain, pH 7.5, 25°C Candida albicans
2.6.1.16 0.34
-
 L-glutamate reaction mixture contains 10 mM L-glutamate, 1 mM EDTA, 1 mM DTT and an appropriately diluted enzyme preparation in 20 mM HEPES buffer (pH 7.5) at 25°C Candida albicans
2.6.1.16 0.34
-
 L-glutamine glutamine 6-phosphate-synthetic activity, wild-type, pH 7.5, 25°C Candida albicans
2.6.1.16 0.64
-
 L-gamma-glutamyl-p-nitroanilide wild-type with C-terminal His-tag, pH 7.5, 25°C Candida albicans
2.6.1.16 0.67
-
 L-gamma-glutamyl-p-nitroanilide wild-type, pH 7.5, 25°C Candida albicans
2.6.1.16 0.67
-
 L-gamma -glutamyl-p-nitroanilide reaction mixture contains 1 mM L-gamma-glutamyl-p-nitroanilide, 1 mM EDTA, 1 mM DTT and the appropriately diluted enzyme preparation in 20 mM HEPES buffer (pH 7.5) at 25°C Candida albicans
2.6.1.16 1.08
-
 D-fructose 6-phosphate hexose phosphate-isomerizing activity, wild-type with N-terminal His-tag, pH 7.5, 25°C Candida albicans
2.6.1.16 1.2
-
 D-fructose 6-phosphate reaction mixture contains 2 mM D-fructose 6-phosphate, 0.5 mM NADP+, 1 mM EDTA, and appropriately diluted GlcN-6-P synthase preparation in 50 mM Tris/HCl buffer (pH 7.5) at 25°C Candida albicans
2.6.1.16 1.2
-
 D-fructose 6-phosphate hexose phosphate-isomerizing activity, wild-type, pH 7.5, 25°C Candida albicans
2.6.1.16 1.4
-
 D-fructose 6-phosphate hexose phosphate-isomerizing activity, residues 346-712, His-tagged hexose phosphate-isomerizing domain, pH 7.5, 25°C Candida albicans
2.6.1.16 1.41
-
 D-fructose 6-phosphate glutamine 6-phosphate-synthetic activity, wild-type, pH 7.5, 25°C Candida albicans
2.6.1.16 6.5
-
 D-fructose 6-phosphate hexose phosphate-isomerizing activity, wild-type with C-terminal His-tag, pH 7.5, 25°C Candida albicans
2.6.1.16 12.2
-
 L-gamma-glutamyl-p-nitroanilide wild-type with N-terminal His-tag, pH 7.5, 25°C Candida albicans

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
2.6.1.16 39500
-
 4 * 39500, hexose phosphate-isomerizing domain, Superdex 200 HR 10/30 gel filtration Candida albicans
2.6.1.16 41000
-
 glutamine amide-hydrolysing domain, SDS-PAGE Candida albicans
2.6.1.16 42000
-
 1 * 42000, glutamine amide-hydrolysing domain, Superdex 200 HR 10/30 gel filtration Candida albicans
2.6.1.16 159000
-
 hexose phosphate-isomerizing domain, Superdex 200 HR 10/30 gel filtration Candida albicans

Organism

EC Number Organism UniProt Comment Textmining
2.6.1.16 Candida albicans
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.6.1.16 Ni2+-IDA agarose affinity chromatography and HiTrap desalting column chromatography Candida albicans

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
2.6.1.16 0.021
-
 glutamine 6-phosphate-synthetic activity, mutant V711F, pH 7.5, 25°C Candida albicans
2.6.1.16 0.05
-
 glutamine 6-phosphate-synthetic activity, mutant W97G, pH 7.5, 25°C Candida albicans
2.6.1.16 0.095
-
 amidohydrolysing activity of the mutant enzyme W97G using D-fructose 6-phosphate as substrate Candida albicans
2.6.1.16 0.256
-
 amidohydrolysing activity of the mutant enzyme W97F using D-fructose 6-phosphate as substrate Candida albicans
2.6.1.16 0.89
-
 amidohydrolysing activity of the wild type enzyme using D-fructose 6-phosphate as substrate Candida albicans
2.6.1.16 0.94
-
 amidohydrolysing activity of the mutant enzyme V711F using D-fructose 6-phosphate as substrate Candida albicans
2.6.1.16 1.2
-
 glutamine 6-phosphate-synthetic activity, mutant W97F, pH 7.5, 25°C Candida albicans
2.6.1.16 12
-
 glutamine 6-phosphate-synthetic activity, wild-type, pH 7.5, 25°C Candida albicans

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.6.1.16 D-fructose 6-phosphate determination of hexose phosphate-isomerizing activity Candida albicans D-glucose 6-phosphate
-
 ?
2.6.1.16 L-gamma-glutamyl-p-nitroanilide + D-fructose 6-phosphate
-
 Candida albicans ?
-
 ?
2.6.1.16 L-gamma-glutamyl-p-nitroanilide + H2O determination of amidohydrolysing activity Candida albicans L-glutamine + p-nitroaniline
-
 ?
2.6.1.16 L-glutamine + D-fructose 6-phosphate
-
 Candida albicans L-glutamate + D-glucosamine 6-phosphate
-
 ?

Subunits

EC Number Subunits Comment Organism
2.6.1.16 homotetramer 4 * 39500, hexose phosphate-isomerizing domain, Superdex 200 HR 10/30 gel filtration Candida albicans
2.6.1.16 monomer 1 * 42000, glutamine amide-hydrolysing domain, Superdex 200 HR 10/30 gel filtration Candida albicans

Synonyms

EC Number Synonyms Comment Organism
2.6.1.16 Gfa1p
-
 Candida albicans
2.6.1.16 glucosamine-6-phosphate synthase
-
 Candida albicans
2.6.1.16 L-glutamine:L-fructose-6-phosphate amidotransferase
-
 Candida albicans

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.6.1.16 0.00092
-
 L-gamma-glutamyl-p-nitroanilide wild-type with N-terminal His-tag, pH 7.5, 25°C Candida albicans
2.6.1.16 0.004
-
 D-fructose 6-phosphate hexose phosphate-isomerizing activity, wild-type with C-terminal His-tag, pH 7.5, 25°C Candida albicans
2.6.1.16 0.0075
-
 L-gamma-glutamyl-p-nitroanilide residues 1-345, His-tagged glutamine amide-hydrolysing domain, pH 7.5, 25°C Candida albicans
2.6.1.16 0.0092
-
 D-fructose 6-phosphate hexose phosphate-isomerizing activity, residues 346-712, His-tagged hexose phosphate-isomerizing domain, pH 7.5, 25°C Candida albicans
2.6.1.16 0.012
-
 L-gamma-glutamyl-p-nitroanilide wild-type, pH 7.5, 25°C Candida albicans
2.6.1.16 0.0123
-
 L-gamma-glutamyl-p-nitroanilide reaction mixture contains 1 mM L-gamma-glutamyl-p-nitroanilide, 1 mM EDTA, 1 mM DTT and the appropriately diluted enzyme preparation in 20 mM HEPES buffer (pH 7.5) at 25°C Candida albicans
2.6.1.16 0.013
-
 L-gamma-glutamyl-p-nitroanilide wild-type with C-terminal His-tag, pH 7.5, 25°C Candida albicans
2.6.1.16 0.021
-
 D-fructose 6-phosphate hexose phosphate-isomerizing activity, wild-type with N-terminal His-tag, pH 7.5, 25°C Candida albicans
2.6.1.16 0.021
-
 D-fructose 6-phosphate hexose phosphate-isomerizing activity, wild-type, pH 7.5, 25°C Candida albicans
2.6.1.16 0.0213
-
 D-fructose 6-phosphate reaction mixture contains 2 mM D-fructose 6-phosphate, 0.5 mM NADP+, 1 mM EDTA, and appropriately diluted GlcN-6-P synthase preparation in 50 mM Tris/HCl buffer (pH 7.5) at 25°C Candida albicans
2.6.1.16 12
-
 L-glutamate reaction mixture contains 10 mM L-glutamate, 1 mM EDTA, 1 mM DTT and an appropriately diluted enzyme preparation in 20 mM HEPES buffer (pH 7.5) at 25°C Candida albicans
2.6.1.16 12
-
 L-glutamine glutamine 6-phosphate-synthetic activity, wild-type, pH 7.5, 25°C Candida albicans