[103] N-Acetylglucosamine-6-phosphate deacetylase and glucosamine-6-phosphate deaminase from Escherichia coli

White, Richard; Pasternak, C. A.

doi:10.1016/s0076-6879(75)41105-3

Cited by 17 publications

(10 citation statements)

References 13 publications

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“…3b) (30). In one molecule, there is residual difference density suggesting a partial (Ͻ0.3) occupancy of unhydrolyzed substrate, GlcNAc-6-P, and although partial occupancy of substrate sheds light on catalysis (see "Discussion"), it was not refined due to the constraint of limited resolution.…”

Section: Resultsmentioning

confidence: 99%

The Three-dimensional Structure of the N-Acetylglucosamine-6-phosphate Deacetylase, NagA, from Bacillus subtilis

Vincent

Yates

Garman

et al. 2004

Journal of Biological Chemistry

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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.cnrsmrs.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 Å. The structure presents two domains, a (␤/␣) 8 barrel enclosing the active center and a small ␤ 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.

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Section: Resultsmentioning

confidence: 99%

The Three-dimensional Structure of the N-Acetylglucosamine-6-phosphate Deacetylase, NagA, from Bacillus subtilis

Vincent

Yates

Garman

et al. 2004

Journal of Biological Chemistry

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“…GlcNAc-6-P deacetylase NagA of S. oneidensis catalyzing the second step of the NAG pathway, which is broadly conserved in bacteria and eukaryotes, has a K m value (0.43 mM) similar to NagA from E. coli (0.4 mM) (35). The k cat of GlcNAc-6-P deacetylase (12.0 s Ϫ1 ) is comparable with that of GlcNAc kinase (Table 3).…”

Section: Properties Of Individual Enzymesmentioning

confidence: 90%

“…The amount of GlcNAc-6-P consumed after 0 -3 min was determined by using p-dimethylaminobenzaldehyde reagent and reading the absorbance at 585 nm (35). Determination of the k cat and K m values was performed by varying the concentration of GlcNAc-6-P in the range of 0.1-5.0 mM.…”

Section: Enzyme Assaysmentioning

confidence: 99%

“…GlcN-6-P deaminase activity was assayed by coupling the formation of Fru-6-P to the reduction of NADP ϩ to NADPH and detection at 340 nm as described (35). Briefly, 1.5-3.0 g of purified GlcN-6-P deaminase was added to 200 l of reaction mixture containing 40 mM sodium phosphate buffer (pH 7.5), 0.2 mM NADP ϩ , 8 units of phosphoglucose isomerase, 3 units of glucose-6-phosphate dehydrogenase, and 1 mM GlcN-6-P. No activity was detected in a control experiment without phosphoglucose isomerase, which verified the production of Fru-6-P but not glucose 6-phosphate.…”

Section: Enzyme Assaysmentioning

confidence: 99%

See 1 more Smart Citation

Comparative Genomics and Experimental Characterization of N-Acetylglucosamine Utilization Pathway of Shewanella oneidensis

Yang

Rodionov

et al. 2006

Journal of Biological Chemistry

131

154

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We used a comparative genomics approach implemented in the SEED annotation environment to reconstruct the chitin and GlcNAc utilization subsystem and regulatory network in most proteobacteria, including 11 species of Shewanella with completely sequenced genomes. Comparative analysis of candidate regulatory sites allowed us to characterize three different GlcNAc-specific regulons, NagC, NagR, and NagQ, in various proteobacteria and to tentatively assign a number of novel genes with specific functional roles, in particular new GlcNAc-related transport systems, to this subsystem. Genes SO3506 and SO3507, originally annotated as hypothetical in Shewanella oneidensis MR-1, were suggested to encode novel variants of GlcN-6-P deaminase and GlcNAc kinase, respectively. Reconstitution of the GlcNAc catabolic pathway in vitro using these purified recombinant proteins and GlcNAc-6-P deacetylase (SO3505) validated the entire pathway. Kinetic characterization of GlcN-6-P deaminase demonstrated that it is the subject of allosteric activation by GlcNAc-6-P. Consistent with genomic data, all tested Shewanella strains except S. frigidimarina, which lacked representative genes for the GlcNAc metabolism, were capable of utilizing GlcNAc as the sole source of carbon and energy. This study expands the range of carbon substrates utilized by Shewanella spp., unambiguously identifies several genes involved in chitin metabolism, and describes a novel variant of the classical three-step biochemical conversion of GlcNAc to fructose 6-phosphate first described in Escherichia coli.

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“…(ii) GlcN-6-phosphate deaminase GlcN-6-phosphate deaminase was measured in a coupled enzyme assay as described by White & Pasternak (1975). The reaction mixture contained in 0.8 ml: 30 pmol Tris/HCl pH 7.5; 2 pmol NADP; 1.5 units of glucose-6-phosphate dehydrogenase (EC 1.1.1.49); 2 units phosphoglucoisomerase (EC 5.3.1.9); 2.0 pmol GlcN-&phosphate and cell-free extract.…”

Section: Organism Candida Albicansmentioning

confidence: 99%

Enzymes of N-Acetylglucosamine Metabolism during Germ-tube Formation in Candida albicans

1982

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The enzymes of N-acetyl-D-glucosamine (GlcNAc) metabolism, GlcNAc-6-phosphate deacetylase and GlcN-6-phosphate deaminase were found to be inducible in Candida albicans. The pattern of induction for these enzymes was the same under conditions of germ-tube formation (37 degrees C) and where yeast cells metabolized GlcNAc with no change in morphology (28 degrees C); this indicates that these enzymes are not control points in the dimorphic development of C. albicans. During induction there was a 40-and 25-fold increase in specific activity for the deacetylase and the deaminase, respectively, and the maximum specific activity correspond to the time when all the GlcNAc had been metabolized. The presence of lomofungin (an inhibitor of transcription) or trichodermin (an inhibitor of translation) in cell suspensions of C. albicans containing GlcNAc prevented the increase in specific activity of these enzymes. 2-Deoxyglucose inhibited germ-tube formation, partially inhibited the induction of the deacetylase (43%) and the deaminase (60%), but did not affect the growth of C. albicans on either Glc or GlcNAc. GlcN-6-phosphate was a competitive inhibitor of the deacetylase with a Ki of 1.4 mM while the other product of the reaction, acetate, did not inhibit the enzyme. The Km value for GlcN-6-phosphate on GlcN-6-phosphate deaminase was 0.24 mM. Incubation of starved yeast cells with GlcNAc produced a four-fold increase in the specific activity of UDP-GlcNAc-pyrophosphorylase at either 28 degrees C or 37 degrees C.

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[103] N-Acetylglucosamine-6-phosphate deacetylase and glucosamine-6-phosphate deaminase from Escherichia coli

Cited by 17 publications

References 13 publications

The Three-dimensional Structure of the N-Acetylglucosamine-6-phosphate Deacetylase, NagA, from Bacillus subtilis

The Three-dimensional Structure of the N-Acetylglucosamine-6-phosphate Deacetylase, NagA, from Bacillus subtilis

Comparative Genomics and Experimental Characterization of N-Acetylglucosamine Utilization Pathway of Shewanella oneidensis

Enzymes of N-Acetylglucosamine Metabolism during Germ-tube Formation in Candida albicans

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