the backward direction. The reverse reaction is strongly unfavored and is probably physiologically insignificant, but it was useful for obtaining a better kinetic description of the enzyme. A sequential mechanism, with ordered release of products and a slow isomerization of the enzyme-acetate complex, is proposed. This model is supported by data from substrate and product inhibition patterns in both directions of the reaction. << Less

Arch. Biochem. Biophys. 340:338-346(1997) [PubMed] [EuropePMC]

  • The three-dimensional structure of the N-acetylglucosamine-6-phosphate deacetylase, NagA, from Bacillus subtilis: a member of the urease superfamily.

    Vincent F., Yates D., Garman E., Davies G.J., Brannigan J.A.

    The enzyme N-acetylglucosamine-6-phosphate deacetylase, NagA, catalyzes the hydrolysis of the N-acetyl group of GlcNAc-6-P to yield glucosamine 6-phosphate and acetate, the first committed step in the biosynthetic pathway to amino-sugar-nucleotides. It is classified into carbohydrate esterase fami ... >> More

    The enzyme N-acetylglucosamine-6-phosphate deacetylase, NagA, catalyzes the hydrolysis of the N-acetyl group of GlcNAc-6-P to yield glucosamine 6-phosphate and acetate, the first committed step in the biosynthetic pathway to amino-sugar-nucleotides. It is classified into carbohydrate esterase family CE-9 (see afmb.cnrs-mrs.fr/CAZY/). Here we report the cloning, expression, and three-dimensional structure (Protein Data Bank code 1un7) determination by x-ray crystallography of the Bacillus subtilis NagA at a resolution of 2.0 A. The structure presents two domains, a (beta/alpha)(8) barrel enclosing the active center and a small beta barrel domain. The structure is dimeric, and the substrate phosphate coordination at the active center is provided by an Arg/His pair contributed from the second molecule of the dimer. Both the overall structure and the active center bear a striking similarity to the urease superfamily with two metals involved in substrate binding and catalysis. PIXE (Proton-Induced x-ray Emission) data show that iron is the predominant metal in the purified protein. We propose a catalytic mechanism involving proton donation to the leaving group by aspartate, nucleophilic attack by an Fe-bridged hydroxide, and stabilization of the carbonyl oxygen by one of the two Fe atoms of the pair. We believe that this is the first sugar deacetylase to utilize this fold and catalytic mechanism. << Less

    J. Biol. Chem. 279:2809-2816(2004) [PubMed] [EuropePMC]

  • Structural analysis of N-acetylglucosamine-6-phosphate deacetylase apoenzyme from Escherichia coli.

    Ferreira F.M., Mendoza-Hernandez G., Castaneda-Bueno M., Aparicio R., Fischer H., Calcagno M.L., Oliva G.

    We report the crystal structure of the apoenzyme of N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase from Escherichia coli (EcNAGPase) and the spectrometric evidence of the presence of Zn2+ in the native protein. The GlcNAc6P deacetylase is an enzyme of the amino sugar catabolic pathway that ... >> More

    We report the crystal structure of the apoenzyme of N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase from Escherichia coli (EcNAGPase) and the spectrometric evidence of the presence of Zn2+ in the native protein. The GlcNAc6P deacetylase is an enzyme of the amino sugar catabolic pathway that catalyzes the conversion of the GlcNAc6P into glucosamine 6-phosphate (GlcN6P). The crystal structure was phased by the single isomorphous replacement with anomalous scattering (SIRAS) method using low-resolution (2.9 A) iodine anomalous scattering and it was refined against a native dataset up to 2.0 A resolution. The structure is similar to two other NAGPases whose structures are known from Thermotoga maritima (TmNAGPase) and Bacillus subtilis (BsNAGPase); however, it shows a phosphate ion bound at the metal-binding site. Compared to these previous structures, the apoenzyme shows extensive conformational changes in two loops adjacent to the active site. The E. coli enzyme is a tetramer and its dimer-dimer interface was analyzed. The tetrameric structure was confirmed in solution by small-angle X-ray scattering data. Although no metal ions were detected in the present structure, experiments of photon-induced X-ray emission (PIXE) spectra and of inductively coupled plasma emission spectroscopy (ICP-AES) with enzyme that was neither exposed to chelating agents nor metal ions during purification, revealed the presence of 1.4 atoms of Zn per polypeptide chain. Enzyme inactivation by metal-sequestering agents and subsequent reactivation by the addition of several divalent cations, demonstrate the role of metal ions in EcNAGPase structure and catalysis. << Less

    J. Mol. Biol. 359:308-321(2006) [PubMed] [EuropePMC]

    • An enzyme that deacetylates N-acetylglucosamine to glucosamine from Vibrio cholerae non-O1 was purified to homogeneity by sequential procedures. The native enzyme had a molecular mass of 190,000 Da and was predicted to be composed of four identical subunits with molecular masses of 45,000 Da. The ... >> More

    An enzyme that deacetylates N-acetylglucosamine to glucosamine from Vibrio cholerae non-O1 was purified to homogeneity by sequential procedures. The native enzyme had a molecular mass of 190,000 Da and was predicted to be composed of four identical subunits with molecular masses of 45,000 Da. The purified enzyme hydrolyzed N-acetylglucosamine, N-acetylglucosamine 6-phosphate, and N-acetylglucosamine 6-sulfate, but not chitin oligosaccharides, and N-acetylgalactosamine. The deacetylase activity was completely abolished by N-ethylmaleimide, p-chloromercuribenzoate, EDTA, and Cu2+. On the other hand, the activity was activated by Co2+. The amino-terminal amino acids of the purified enzyme were sequenced. Among the 22 N-terminal amino acid residues, 12 residues of Vibrio deacetylase were identical with that of Escherichia coli GlcNAc 6-phosphate deacetylase. << Less

    Biosci Biotechnol Biochem 60:1320-1323(1996) [PubMed] [EuropePMC]

  • The purification and properties of N-acetylglucosamine 6-phosphate deacetylase from Escherichia coli.

    White R.J., Pasternak C.A.

    1. N-Acetylglucosamine 6-phosphate deacetylase and 2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating) (EC 5.3.1.10, glucosamine 6-phosphate deaminase) of Escherichia coliK(12) have been separated by chromatography on DEAE-cellulose. 2. N-Acetylglucosamine 6-phosphate deacetylase h ... >> More

    1. N-Acetylglucosamine 6-phosphate deacetylase and 2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating) (EC 5.3.1.10, glucosamine 6-phosphate deaminase) of Escherichia coliK(12) have been separated by chromatography on DEAE-cellulose. 2. N-Acetylglucosamine 6-phosphate deacetylase has optimum pH8.5 and K(m) 0.8mm. Glucosamine 6-phosphate is a product of the reaction. There appear to be no essential cofactors. Glucosamine 6-phosphate and fructose 6-phosphate inhibit deacetylation. 3. Glucosamine 6-phosphate deaminase has optimum pH7.0 and K(m) 9.0mm. It is stimulated by N-acetylglucosamine 6-phosphate. 4. We propose that the deacetylase be termed 2-acetamido-2-deoxy-d-glucose 6-phosphate amidohydrolase (EC 3.5.1.-), with acetylglucosamine 6-phosphate deacetylase as a trivial name. << Less

    Biochem. J. 105:121-125(1967) [PubMed] [EuropePMC]

  • N-acetylglucosamine-6-phosphate deacetylase from Escherichia coli: purification and molecular and kinetic characterization.

    Souza J.M., Plumbridge J.A., Calcagno M.L.

    N-Acetylglucosamine-6-phosphate deacetylase (E.C. 3.5.1.25), an enzyme of the amino sugar utilization pathway, has been purified from an overproducing strain of Escherichia coli. The enzyme is a tetramer of identical 41-kDa subunits. The sedimentation coefficient of the oligomer is 6.5 s(20),w and ... >> More

    N-Acetylglucosamine-6-phosphate deacetylase (E.C. 3.5.1.25), an enzyme of the amino sugar utilization pathway, has been purified from an overproducing strain of Escherichia coli. The enzyme is a tetramer of identical 41-kDa subunits. The sedimentation coefficient of the oligomer is 6.5 s(20),w and it has a pI of 4.9. The circular dichroism spectrum of the enzyme in the far uv range suggests that it is a protein belonging to the alpha/beta structural family. In the native enzyme, two thiols per chain are titrated with 5-5'-dithio-bis(2-nitrobenzoate) (NbS2); one reacts rapidly, the other more slowly. The reaction of the more reactive sulfhydryl completely inhibits the activity of the enzyme. Three thiols, of the total of eight per subunit of the native enzyme, are modified by methyl iodide without significantly changing the kinetic parameters; the methylated enzyme becomes insensitive to NbS2 inhibition. One of the enzyme reaction products, glucosamine 6-phosphate, completely protects this thiol from NbS2 reaction. The kinetics of the deacetylase reaction have been studied both in the forward direction and in the backward direction. The reverse reaction is strongly unfavored and is probably physiologically insignificant, but it was useful for obtaining a better kinetic description of the enzyme. A sequential mechanism, with ordered release of products and a slow isomerization of the enzyme-acetate complex, is proposed. This model is supported by data from substrate and product inhibition patterns in both directions of the reaction. << Less

    Arch. Biochem. Biophys. 340:338-346(1997) [PubMed] [EuropePMC]

  • The three-dimensional structure of the N-acetylglucosamine-6-phosphate deacetylase, NagA, from Bacillus subtilis: a member of the urease superfamily.

    Vincent F., Yates D., Garman E., Davies G.J., Brannigan J.A.

    The enzyme N-acetylglucosamine-6-phosphate deacetylase, NagA, catalyzes the hydrolysis of the N-acetyl group of GlcNAc-6-P to yield glucosamine 6-phosphate and acetate, the first committed step in the biosynthetic pathway to amino-sugar-nucleotides. It is classified into carbohydrate esterase fami ... >> More

    The enzyme N-acetylglucosamine-6-phosphate deacetylase, NagA, catalyzes the hydrolysis of the N-acetyl group of GlcNAc-6-P to yield glucosamine 6-phosphate and acetate, the first committed step in the biosynthetic pathway to amino-sugar-nucleotides. It is classified into carbohydrate esterase family CE-9 (see afmb.cnrs-mrs.fr/CAZY/). Here we report the cloning, expression, and three-dimensional structure (Protein Data Bank code 1un7) determination by x-ray crystallography of the Bacillus subtilis NagA at a resolution of 2.0 A. The structure presents two domains, a (beta/alpha)(8) barrel enclosing the active center and a small beta barrel domain. The structure is dimeric, and the substrate phosphate coordination at the active center is provided by an Arg/His pair contributed from the second molecule of the dimer. Both the overall structure and the active center bear a striking similarity to the urease superfamily with two metals involved in substrate binding and catalysis. PIXE (Proton-Induced x-ray Emission) data show that iron is the predominant metal in the purified protein. We propose a catalytic mechanism involving proton donation to the leaving group by aspartate, nucleophilic attack by an Fe-bridged hydroxide, and stabilization of the carbonyl oxygen by one of the two Fe atoms of the pair. We believe that this is the first sugar deacetylase to utilize this fold and catalytic mechanism. << Less

    J. Biol. Chem. 279:2809-2816(2004) [PubMed] [EuropePMC]

  • Structural analysis of N-acetylglucosamine-6-phosphate deacetylase apoenzyme from Escherichia coli.

    Ferreira F.M., Mendoza-Hernandez G., Castaneda-Bueno M., Aparicio R., Fischer H., Calcagno M.L., Oliva G.

    We report the crystal structure of the apoenzyme of N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase from Escherichia coli (EcNAGPase) and the spectrometric evidence of the presence of Zn2+ in the native protein. The GlcNAc6P deacetylase is an enzyme of the amino sugar catabolic pathway that ... >> More

    We report the crystal structure of the apoenzyme of N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase from Escherichia coli (EcNAGPase) and the spectrometric evidence of the presence of Zn2+ in the native protein. The GlcNAc6P deacetylase is an enzyme of the amino sugar catabolic pathway that catalyzes the conversion of the GlcNAc6P into glucosamine 6-phosphate (GlcN6P). The crystal structure was phased by the single isomorphous replacement with anomalous scattering (SIRAS) method using low-resolution (2.9 A) iodine anomalous scattering and it was refined against a native dataset up to 2.0 A resolution. The structure is similar to two other NAGPases whose structures are known from Thermotoga maritima (TmNAGPase) and Bacillus subtilis (BsNAGPase); however, it shows a phosphate ion bound at the metal-binding site. Compared to these previous structures, the apoenzyme shows extensive conformational changes in two loops adjacent to the active site. The E. coli enzyme is a tetramer and its dimer-dimer interface was analyzed. The tetrameric structure was confirmed in solution by small-angle X-ray scattering data. Although no metal ions were detected in the present structure, experiments of photon-induced X-ray emission (PIXE) spectra and of inductively coupled plasma emission spectroscopy (ICP-AES) with enzyme that was neither exposed to chelating agents nor metal ions during purification, revealed the presence of 1.4 atoms of Zn per polypeptide chain. Enzyme inactivation by metal-sequestering agents and subsequent reactivation by the addition of several divalent cations, demonstrate the role of metal ions in EcNAGPase structure and catalysis. << Less

    J. Mol. Biol. 359:308-321(2006) [PubMed] [EuropePMC]

    • An enzyme that deacetylates N-acetylglucosamine to glucosamine from Vibrio cholerae non-O1 was purified to homogeneity by sequential procedures. The native enzyme had a molecular mass of 190,000 Da and was predicted to be composed of four identical subunits with molecular masses of 45,000 Da. The ... >> More

    An enzyme that deacetylates N-acetylglucosamine to glucosamine from Vibrio cholerae non-O1 was purified to homogeneity by sequential procedures. The native enzyme had a molecular mass of 190,000 Da and was predicted to be composed of four identical subunits with molecular masses of 45,000 Da. The purified enzyme hydrolyzed N-acetylglucosamine, N-acetylglucosamine 6-phosphate, and N-acetylglucosamine 6-sulfate, but not chitin oligosaccharides, and N-acetylgalactosamine. The deacetylase activity was completely abolished by N-ethylmaleimide, p-chloromercuribenzoate, EDTA, and Cu2+. On the other hand, the activity was activated by Co2+. The amino-terminal amino acids of the purified enzyme were sequenced. Among the 22 N-terminal amino acid residues, 12 residues of Vibrio deacetylase were identical with that of Escherichia coli GlcNAc 6-phosphate deacetylase. << Less

    Biosci Biotechnol Biochem 60:1320-1323(1996) [PubMed] [EuropePMC]

  • The purification and properties of N-acetylglucosamine 6-phosphate deacetylase from Escherichia coli.

    White R.J., Pasternak C.A.

    1. N-Acetylglucosamine 6-phosphate deacetylase and 2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating) (EC 5.3.1.10, glucosamine 6-phosphate deaminase) of Escherichia coliK(12) have been separated by chromatography on DEAE-cellulose. 2. N-Acetylglucosamine 6-phosphate deacetylase h ... >> More

    1. N-Acetylglucosamine 6-phosphate deacetylase and 2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating) (EC 5.3.1.10, glucosamine 6-phosphate deaminase) of Escherichia coliK(12) have been separated by chromatography on DEAE-cellulose. 2. N-Acetylglucosamine 6-phosphate deacetylase has optimum pH8.5 and K(m) 0.8mm. Glucosamine 6-phosphate is a product of the reaction. There appear to be no essential cofactors. Glucosamine 6-phosphate and fructose 6-phosphate inhibit deacetylation. 3. Glucosamine 6-phosphate deaminase has optimum pH7.0 and K(m) 9.0mm. It is stimulated by N-acetylglucosamine 6-phosphate. 4. We propose that the deacetylase be termed 2-acetamido-2-deoxy-d-glucose 6-phosphate amidohydrolase (EC 3.5.1.-), with acetylglucosamine 6-phosphate deacetylase as a trivial name. << Less

    Biochem. J. 105:121-125(1967) [PubMed] [EuropePMC]

  • N-acetylglucosamine-6-phosphate deacetylase from Escherichia coli: purification and molecular and kinetic characterization.

    Souza J.M., Plumbridge J.A., Calcagno M.L.

    N-Acetylglucosamine-6-phosphate deacetylase (E.C. 3.5.1.25), an enzyme of the amino sugar utilization pathway, has been purified from an overproducing strain of Escherichia coli. The enzyme is a tetramer of identical 41-kDa subunits. The sedimentation coefficient of the oligomer is 6.5 s(20),w and ... >> More

    N-Acetylglucosamine-6-phosphate deacetylase (E.C. 3.5.1.25), an enzyme of the amino sugar utilization pathway, has been purified from an overproducing strain of Escherichia coli. The enzyme is a tetramer of identical 41-kDa subunits. The sedimentation coefficient of the oligomer is 6.5 s(20),w and it has a pI of 4.9. The circular dichroism spectrum of the enzyme in the far uv range suggests that it is a protein belonging to the alpha/beta structural family. In the native enzyme, two thiols per chain are titrated with 5-5'-dithio-bis(2-nitrobenzoate) (NbS2); one reacts rapidly, the other more slowly. The reaction of the more reactive sulfhydryl completely inhibits the activity of the enzyme. Three thiols, of the total of eight per subunit of the native enzyme, are modified by methyl iodide without significantly changing the kinetic parameters; the methylated enzyme becomes insensitive to NbS2 inhibition. One of the enzyme reaction products, glucosamine 6-phosphate, completely protects this thiol from NbS2 reaction. The kinetics of the deacetylase reaction have been studied both in the forward direction and in the backward direction. The reverse reaction is strongly unfavored and is probably physiologically insignificant, but it was useful for obtaining a better kinetic description of the enzyme. A sequential mechanism, with ordered release of products and a slow isomerization of the enzyme-acetate complex, is proposed. This model is supported by data from substrate and product inhibition patterns in both directions of the reaction. << Less

    Arch. Biochem. Biophys. 340:338-346(1997) [PubMed] [EuropePMC]

  • The three-dimensional structure of the N-acetylglucosamine-6-phosphate deacetylase, NagA, from Bacillus subtilis: a member of the urease superfamily.

    Vincent F., Yates D., Garman E., Davies G.J., Brannigan J.A.

    The enzyme N-acetylglucosamine-6-phosphate deacetylase, NagA, catalyzes the hydrolysis of the N-acetyl group of GlcNAc-6-P to yield glucosamine 6-phosphate and acetate, the first committed step in the biosynthetic pathway to amino-sugar-nucleotides. It is classified into carbohydrate esterase fami ... >> More

    The enzyme N-acetylglucosamine-6-phosphate deacetylase, NagA, catalyzes the hydrolysis of the N-acetyl group of GlcNAc-6-P to yield glucosamine 6-phosphate and acetate, the first committed step in the biosynthetic pathway to amino-sugar-nucleotides. It is classified into carbohydrate esterase family CE-9 (see afmb.cnrs-mrs.fr/CAZY/). Here we report the cloning, expression, and three-dimensional structure (Protein Data Bank code 1un7) determination by x-ray crystallography of the Bacillus subtilis NagA at a resolution of 2.0 A. The structure presents two domains, a (beta/alpha)(8) barrel enclosing the active center and a small beta barrel domain. The structure is dimeric, and the substrate phosphate coordination at the active center is provided by an Arg/His pair contributed from the second molecule of the dimer. Both the overall structure and the active center bear a striking similarity to the urease superfamily with two metals involved in substrate binding and catalysis. PIXE (Proton-Induced x-ray Emission) data show that iron is the predominant metal in the purified protein. We propose a catalytic mechanism involving proton donation to the leaving group by aspartate, nucleophilic attack by an Fe-bridged hydroxide, and stabilization of the carbonyl oxygen by one of the two Fe atoms of the pair. We believe that this is the first sugar deacetylase to utilize this fold and catalytic mechanism. << Less

    J. Biol. Chem. 279:2809-2816(2004) [PubMed] [EuropePMC]

  • Structural analysis of N-acetylglucosamine-6-phosphate deacetylase apoenzyme from Escherichia coli.

    Ferreira F.M., Mendoza-Hernandez G., Castaneda-Bueno M., Aparicio R., Fischer H., Calcagno M.L., Oliva G.

    We report the crystal structure of the apoenzyme of N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase from Escherichia coli (EcNAGPase) and the spectrometric evidence of the presence of Zn2+ in the native protein. The GlcNAc6P deacetylase is an enzyme of the amino sugar catabolic pathway that ... >> More

    We report the crystal structure of the apoenzyme of N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase from Escherichia coli (EcNAGPase) and the spectrometric evidence of the presence of Zn2+ in the native protein. The GlcNAc6P deacetylase is an enzyme of the amino sugar catabolic pathway that catalyzes the conversion of the GlcNAc6P into glucosamine 6-phosphate (GlcN6P). The crystal structure was phased by the single isomorphous replacement with anomalous scattering (SIRAS) method using low-resolution (2.9 A) iodine anomalous scattering and it was refined against a native dataset up to 2.0 A resolution. The structure is similar to two other NAGPases whose structures are known from Thermotoga maritima (TmNAGPase) and Bacillus subtilis (BsNAGPase); however, it shows a phosphate ion bound at the metal-binding site. Compared to these previous structures, the apoenzyme shows extensive conformational changes in two loops adjacent to the active site. The E. coli enzyme is a tetramer and its dimer-dimer interface was analyzed. The tetrameric structure was confirmed in solution by small-angle X-ray scattering data. Although no metal ions were detected in the present structure, experiments of photon-induced X-ray emission (PIXE) spectra and of inductively coupled plasma emission spectroscopy (ICP-AES) with enzyme that was neither exposed to chelating agents nor metal ions during purification, revealed the presence of 1.4 atoms of Zn per polypeptide chain. Enzyme inactivation by metal-sequestering agents and subsequent reactivation by the addition of several divalent cations, demonstrate the role of metal ions in EcNAGPase structure and catalysis. << Less

    J. Mol. Biol. 359:308-321(2006) [PubMed] [EuropePMC]

    • An enzyme that deacetylates N-acetylglucosamine to glucosamine from Vibrio cholerae non-O1 was purified to homogeneity by sequential procedures. The native enzyme had a molecular mass of 190,000 Da and was predicted to be composed of four identical subunits with molecular masses of 45,000 Da. The ... >> More

    An enzyme that deacetylates N-acetylglucosamine to glucosamine from Vibrio cholerae non-O1 was purified to homogeneity by sequential procedures. The native enzyme had a molecular mass of 190,000 Da and was predicted to be composed of four identical subunits with molecular masses of 45,000 Da. The purified enzyme hydrolyzed N-acetylglucosamine, N-acetylglucosamine 6-phosphate, and N-acetylglucosamine 6-sulfate, but not chitin oligosaccharides, and N-acetylgalactosamine. The deacetylase activity was completely abolished by N-ethylmaleimide, p-chloromercuribenzoate, EDTA, and Cu2+. On the other hand, the activity was activated by Co2+. The amino-terminal amino acids of the purified enzyme were sequenced. Among the 22 N-terminal amino acid residues, 12 residues of Vibrio deacetylase were identical with that of Escherichia coli GlcNAc 6-phosphate deacetylase. << Less

    Biosci Biotechnol Biochem 60:1320-1323(1996) [PubMed] [EuropePMC]

  • The purification and properties of N-acetylglucosamine 6-phosphate deacetylase from Escherichia coli.

    White R.J., Pasternak C.A.

    1. N-Acetylglucosamine 6-phosphate deacetylase and 2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating) (EC 5.3.1.10, glucosamine 6-phosphate deaminase) of Escherichia coliK(12) have been separated by chromatography on DEAE-cellulose. 2. N-Acetylglucosamine 6-phosphate deacetylase h ... >> More

    1. N-Acetylglucosamine 6-phosphate deacetylase and 2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating) (EC 5.3.1.10, glucosamine 6-phosphate deaminase) of Escherichia coliK(12) have been separated by chromatography on DEAE-cellulose. 2. N-Acetylglucosamine 6-phosphate deacetylase has optimum pH8.5 and K(m) 0.8mm. Glucosamine 6-phosphate is a product of the reaction. There appear to be no essential cofactors. Glucosamine 6-phosphate and fructose 6-phosphate inhibit deacetylation. 3. Glucosamine 6-phosphate deaminase has optimum pH7.0 and K(m) 9.0mm. It is stimulated by N-acetylglucosamine 6-phosphate. 4. We propose that the deacetylase be termed 2-acetamido-2-deoxy-d-glucose 6-phosphate amidohydrolase (EC 3.5.1.-), with acetylglucosamine 6-phosphate deacetylase as a trivial name. << Less

    Biochem. J. 105:121-125(1967) [PubMed] [EuropePMC]

  • N-acetylglucosamine-6-phosphate deacetylase from Escherichia coli: purification and molecular and kinetic characterization.

    Souza J.M., Plumbridge J.A., Calcagno M.L.

    N-Acetylglucosamine-6-phosphate deacetylase (E.C. 3.5.1.25), an enzyme of the amino sugar utilization pathway, has been purified from an overproducing strain of Escherichia coli. The enzyme is a tetramer of identical 41-kDa subunits. The sedimentation coefficient of the oligomer is 6.5 s(20),w and ... >> More

    N-Acetylglucosamine-6-phosphate deacetylase (E.C. 3.5.1.25), an enzyme of the amino sugar utilization pathway, has been purified from an overproducing strain of Escherichia coli. The enzyme is a tetramer of identical 41-kDa subunits. The sedimentation coefficient of the oligomer is 6.5 s(20),w and it has a pI of 4.9. The circular dichroism spectrum of the enzyme in the far uv range suggests that it is a protein belonging to the alpha/beta structural family. In the native enzyme, two thiols per chain are titrated with 5-5'-dithio-bis(2-nitrobenzoate) (NbS2); one reacts rapidly, the other more slowly. The reaction of the more reactive sulfhydryl completely inhibits the activity of the enzyme. Three thiols, of the total of eight per subunit of the native enzyme, are modified by methyl iodide without significantly changing the kinetic parameters; the methylated enzyme becomes insensitive to NbS2 inhibition. One of the enzyme reaction products, glucosamine 6-phosphate, completely protects this thiol from NbS2 reaction. The kinetics of the deacetylase reaction have been studied both in the forward direction and in the backward direction. The reverse reaction is strongly unfavored and is probably physiologically insignificant, but it was useful for obtaining a better kinetic description of the enzyme. A sequential mechanism, with ordered release of products and a slow isomerization of the enzyme-acetate complex, is proposed. This model is supported by data from substrate and product inhibition patterns in both directions of the reaction. << Less

    Arch. Biochem. Biophys. 340:338-346(1997) [PubMed] [EuropePMC]

  • The three-dimensional structure of the N-acetylglucosamine-6-phosphate deacetylase, NagA, from Bacillus subtilis: a member of the urease superfamily.

    Vincent F., Yates D., Garman E., Davies G.J., Brannigan J.A.

    The enzyme N-acetylglucosamine-6-phosphate deacetylase, NagA, catalyzes the hydrolysis of the N-acetyl group of GlcNAc-6-P to yield glucosamine 6-phosphate and acetate, the first committed step in the biosynthetic pathway to amino-sugar-nucleotides. It is classified into carbohydrate esterase fami ... >> More

    The enzyme N-acetylglucosamine-6-phosphate deacetylase, NagA, catalyzes the hydrolysis of the N-acetyl group of GlcNAc-6-P to yield glucosamine 6-phosphate and acetate, the first committed step in the biosynthetic pathway to amino-sugar-nucleotides. It is classified into carbohydrate esterase family CE-9 (see afmb.cnrs-mrs.fr/CAZY/). Here we report the cloning, expression, and three-dimensional structure (Protein Data Bank code 1un7) determination by x-ray crystallography of the Bacillus subtilis NagA at a resolution of 2.0 A. The structure presents two domains, a (beta/alpha)(8) barrel enclosing the active center and a small beta barrel domain. The structure is dimeric, and the substrate phosphate coordination at the active center is provided by an Arg/His pair contributed from the second molecule of the dimer. Both the overall structure and the active center bear a striking similarity to the urease superfamily with two metals involved in substrate binding and catalysis. PIXE (Proton-Induced x-ray Emission) data show that iron is the predominant metal in the purified protein. We propose a catalytic mechanism involving proton donation to the leaving group by aspartate, nucleophilic attack by an Fe-bridged hydroxide, and stabilization of the carbonyl oxygen by one of the two Fe atoms of the pair. We believe that this is the first sugar deacetylase to utilize this fold and catalytic mechanism. << Less

    J. Biol. Chem. 279:2809-2816(2004) [PubMed] [EuropePMC]

  • Structural analysis of N-acetylglucosamine-6-phosphate deacetylase apoenzyme from Escherichia coli.

    Ferreira F.M., Mendoza-Hernandez G., Castaneda-Bueno M., Aparicio R., Fischer H., Calcagno M.L., Oliva G.

    We report the crystal structure of the apoenzyme of N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase from Escherichia coli (EcNAGPase) and the spectrometric evidence of the presence of Zn2+ in the native protein. The GlcNAc6P deacetylase is an enzyme of the amino sugar catabolic pathway that ... >> More

    We report the crystal structure of the apoenzyme of N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase from Escherichia coli (EcNAGPase) and the spectrometric evidence of the presence of Zn2+ in the native protein. The GlcNAc6P deacetylase is an enzyme of the amino sugar catabolic pathway that catalyzes the conversion of the GlcNAc6P into glucosamine 6-phosphate (GlcN6P). The crystal structure was phased by the single isomorphous replacement with anomalous scattering (SIRAS) method using low-resolution (2.9 A) iodine anomalous scattering and it was refined against a native dataset up to 2.0 A resolution. The structure is similar to two other NAGPases whose structures are known from Thermotoga maritima (TmNAGPase) and Bacillus subtilis (BsNAGPase); however, it shows a phosphate ion bound at the metal-binding site. Compared to these previous structures, the apoenzyme shows extensive conformational changes in two loops adjacent to the active site. The E. coli enzyme is a tetramer and its dimer-dimer interface was analyzed. The tetrameric structure was confirmed in solution by small-angle X-ray scattering data. Although no metal ions were detected in the present structure, experiments of photon-induced X-ray emission (PIXE) spectra and of inductively coupled plasma emission spectroscopy (ICP-AES) with enzyme that was neither exposed to chelating agents nor metal ions during purification, revealed the presence of 1.4 atoms of Zn per polypeptide chain. Enzyme inactivation by metal-sequestering agents and subsequent reactivation by the addition of several divalent cations, demonstrate the role of metal ions in EcNAGPase structure and catalysis. << Less

    J. Mol. Biol. 359:308-321(2006) [PubMed] [EuropePMC]