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Escherichia coli K-12 substr. MG1655 Enzyme: N-acetylglucosamine-6-phosphate deacetylase



Gene: nagA Accession Numbers: EG10632 (EcoCyc), b0677, ECK0665

Regulation Summary Diagram: ?

Subunit composition of N-acetylglucosamine-6-phosphate deacetylase = [NagA]4
         N-acetylglucosamine-6-phosphate deacetylase = NagA

Summary:
N-acetylglucosamine-6-phosphate deacetylase catalyzes the first cytoplasmic reaction in the metabolism of N-acetyl-D-glucosamine. N-acetylglucosamine can serve as the sole source of carbon for E. coli and is transported into the cell via phosphotransferase transport systems. It thus reaches the cytoplasm in the phosphorylated form, N-acetylglucosamine-6-phosphate.

A catalytic mechanism of NagA involving the formation of a tetrahedral intermediate has been proposed [Hall07], and the function of amino acids and the Zn2+ within the active site has been modelled [Hall07a]. The rate-limiting step is the cleavage of the amide bond [Hall07].

Crystal structures of the apoenzyme, the Zn2+-containing enzyme, and a mutant have been solved [Ferreira06, Hall07a]. The enzyme is a homotetramer both in solution [Souza97] and in the crystal structure [Ferreira06].

nagA mutants are unable to grow on N-acetylglucosamine as the sole source of carbon and energy [White68, Holmes72a].

NagA: "N-acetylglucosamine" [Holmes72a]

Review: [Park08]

Gene Citations: [Plumbridge91, Plumbridge89, Vogler89, Rogers88, Plumbridge91a]

Locations: cytosol

Map Position: [700,826 <- 701,974] (15.11 centisomes)
Length: 1149 bp / 382 aa

Molecular Weight of Polypeptide: 40.949 kD (from nucleotide sequence), 41 kD (experimental) [Souza97 ]

Molecular Weight of Multimer: 165 kD (experimental) [Souza97]

pI: 5.96

Unification Links: ASAP:ABE-0002301 , CGSC:464 , EchoBASE:EB0626 , EcoGene:EG10632 , EcoliWiki:b0677 , ModBase:P0AF18 , OU-Microarray:b0677 , PortEco:nagA , PR:PRO_000023336 , Pride:P0AF18 , Protein Model Portal:P0AF18 , RefSeq:NP_415203 , RegulonDB:EG10632 , SMR:P0AF18 , String:511145.b0677 , Swiss-Model:P0AF18 , UniProt:P0AF18

Relationship Links: InterPro:IN-FAMILY:IPR003764 , InterPro:IN-FAMILY:IPR006680 , InterPro:IN-FAMILY:IPR011059 , PDB:Structure:1YMY , PDB:Structure:1YRR , PDB:Structure:2P50 , PDB:Structure:2P53 , Pfam:IN-FAMILY:PF01979

In Paralogous Gene Group: 496 (2 members)

Gene-Reaction Schematic: ?

Genetic Regulation Schematic: ?

GO Terms:

Biological Process: GO:0006046 - N-acetylglucosamine catabolic process Inferred from experiment [Souza97, White67, Hall07a, Hall07, Ferreira06, Holmes72a]
GO:0019262 - N-acetylneuraminate catabolic process Inferred from experiment Inferred by computational analysis [UniProtGOA12, Hopkins13]
GO:0051289 - protein homotetramerization Inferred from experiment [Souza97, Hall07a, Hall07, Ferreira06]
GO:0005975 - carbohydrate metabolic process Inferred by computational analysis [UniProtGOA11a]
GO:0006044 - N-acetylglucosamine metabolic process Inferred by computational analysis [GOA01a]
Molecular Function: GO:0008270 - zinc ion binding Inferred from experiment [Hall07a, Ferreira06, Hall07]
GO:0008448 - N-acetylglucosamine-6-phosphate deacetylase activity Inferred from experiment Inferred by computational analysis [GOA01, GOA01a, White67, Hall07a, Hall07, Ferreira06, Souza97]
GO:0016787 - hydrolase activity Inferred by computational analysis [UniProtGOA11a, GOA01a]
GO:0016810 - hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds Inferred by computational analysis [GOA01a]
GO:0046872 - metal ion binding Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005829 - cytosol Inferred by computational analysis [DiazMejia09]

MultiFun Terms: metabolism central intermediary metabolism amino sugar conversions

Essentiality data for nagA knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enriched Yes 37 Aerobic 6.95   Yes [Gerdes03, Comment 1]
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]
Yes [Feist07, Comment 4]

Credits:
Last-Curated ? 22-Jun-2007 by Keseler I , SRI International


Enzymatic reaction of: N-acetylglucosamine-6-phosphate deacetylase

Synonyms: N-acetyl-D-glucosamine-6-phosphate amidohydrolase

EC Number: 3.5.1.25

N-acetyl-D-glucosamine 6-phosphate + H2O <=> D-glucosamine 6-phosphate + acetate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is physiologically favored in the direction shown.

In Pathways: superpathway of N-acetylglucosamine, N-acetylmannosamine and N-acetylneuraminate degradation , N-acetylglucosamine degradation I , anhydromuropeptides recycling

Summary:
The purified enzyme can catalyze the reverse reaction is measurable in vitro, but it is probably physiologically insignificant [Souza97].

Cofactors or Prosthetic Groups: Zn2+ [Ferreira06, Xu06d]

Inhibitors (Competitive): acetate [Souza97]

Inhibitors (Noncompetitive): D-glucosamine 6-phosphate [Souza97, White67]

Inhibitors (Unknown Mechanism): β-D-fructofuranose 6-phosphate [White67]

Kinetic Parameters:

Substrate
Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
Citations
acetate
20000.0
[Souza97, BRENDA14]
N-acetyl-D-glucosamine 6-phosphate
400.0
[Ferreira00, BRENDA14]
N-acetyl-D-glucosamine 6-phosphate
800.0
[White67, BRENDA14]
N-acetyl-D-glucosamine 6-phosphate
300.0
105.0
[Souza97, BRENDA14]
N-acetyl-D-glucosamine 6-phosphate
100.0, 200.0, 230.0, 640.0, 80.0
35.0, 41.0, 58.0, 92.0, 96.0, 163.0, 177.0
[Hall07, BRENDA14]

pH(opt): 8.5 [White67]


Sequence Features

Feature Class Location Citations Comment
Mutagenesis-Variant 59
[Hall07, UniProt12b]
Alternate sequence: Q → H; UniProt: Large decrease in catalytic activity and substrate affinity, and increase in the average amount of Zn bound to the protein, suggesting that an additional metal ion can bind to this mutant; when associated with H-61.
Mutagenesis-Variant 61
[Hall07, UniProt12b]
Alternate sequence: N → H; UniProt: Large decrease in catalytic activity and substrate affinity, and increase in the average amount of Zn bound to the protein, suggesting that an additional metal ion can bind to this mutant; when associated with H-59.
Mutagenesis-Variant 131
[Hall07, UniProt12b]
Alternate sequence: E → A; UniProt: Large reduction in the amount of the metal cofactor bound to the enzyme.
Alternate sequence: E → Q; UniProt: Large reduction in the amount of the metal cofactor bound to the enzyme.
Metal-Binding-Site 131
[UniProt12a]
UniProt: Zinc.
Amino-Acid-Sites-That-Bind 142
[UniProt12a]
UniProt: Substrate; via amide nitrogen.
Mutagenesis-Variant 143
[Hall07, UniProt12b]
Alternate sequence: H → Q; UniProt: 180-fold decrease in catalytic efficiency.
Alternate sequence: H → N; UniProt: Dramatic decrease in catalytic activity and moderate decrease in substrate affinity, producing a 6000-fold decrease in catalytic efficiency.
Metal-Binding-Site 195
[UniProt12a]
UniProt: Zinc; via tele nitrogen.
Metal-Binding-Site 216
[UniProt12a]
UniProt: Zinc; via tele nitrogen.
Protein-Segment 219 -> 220
[UniProt12a]
UniProt: Substrate binding; Sequence Annotation Type: region of interest.
Amino-Acid-Sites-That-Bind 227
[UniProt12a]
UniProt: Substrate.
Mutagenesis-Variant 251
[Hall07, UniProt12b]
Alternate sequence: H → N; UniProt: 500-fold decrease in catalytic efficiency.
Amino-Acid-Sites-That-Bind 251
[UniProt12a]
UniProt: Substrate; via tele nitrogen.
Mutagenesis-Variant 273
[Hall07, UniProt12b]
Alternate sequence: D → A; UniProt: Loss of catalytic activity.
Alternate sequence: D → N; UniProt: Loss of catalytic activity.
Active-Site 273
[UniProt12a]
UniProt: Proton donor/acceptor.
Protein-Segment 306 -> 308
[UniProt12a]
UniProt: Substrate binding; Sequence Annotation Type: region of interest.


Gene Local Context (not to scale): ?

Transcription Units:

Notes:

History:
10/20/97 Gene b0677 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10632; confirmed by SwissProt match.


References

Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554

BRENDA14: BRENDA team (2014). "Imported from BRENDA version existing on Aug 2014." http://www.brenda-enzymes.org.

DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114

Feist07: Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, Karp PD, Broadbelt LJ, Hatzimanikatis V, Palsson BO (2007). "A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information." Mol Syst Biol 3;121. PMID: 17593909

Ferreira00: Ferreira FM, Mendoza-Hernandez G, Calcagno ML, Minauro F, Delboni LF, Oliva G (2000). "Crystallization and preliminary crystallographic analysis of N-acetylglucosamine 6-phosphate deacetylase from Escherichia coli." Acta Crystallogr D Biol Crystallogr 56(Pt 5);670-2. PMID: 10771446

Ferreira06: Ferreira FM, Mendoza-Hernandez G, Castaneda-Bueno M, Aparicio R, Fischer H, Calcagno ML, Oliva G (2006). "Structural analysis of N-acetylglucosamine-6-phosphate deacetylase apoenzyme from Escherichia coli." J Mol Biol 359(2);308-21. PMID: 16630633

Gerdes03: Gerdes SY, Scholle MD, Campbell JW, Balazsi G, Ravasz E, Daugherty MD, Somera AL, Kyrpides NC, Anderson I, Gelfand MS, Bhattacharya A, Kapatral V, D'Souza M, Baev MV, Grechkin Y, Mseeh F, Fonstein MY, Overbeek R, Barabasi AL, Oltvai ZN, Osterman AL (2003). "Experimental determination and system level analysis of essential genes in Escherichia coli MG1655." J Bacteriol 185(19);5673-84. PMID: 13129938

GOA01: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

GOA01a: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

Hall07: Hall RS, Xiang DF, Xu C, Raushel FM (2007). "N-Acetyl-d-glucosamine-6-phosphate Deacetylase: Substrate Activation via a Single Divalent Metal Ion." Biochemistry 46(27):7942-52. PMID: 17567047

Hall07a: Hall RS, Brown S, Fedorov AA, Fedorov EV, Xu C, Babbitt PC, Almo SC, Raushel FM (2007). "Structural Diversity within the Mononuclear and Binuclear Active Sites of N-Acetyl-d-glucosamine-6-phosphate Deacetylase(,)." Biochemistry 46(27):7953-62. PMID: 17567048

Holmes72a: Holmes RP, Russell RR (1972). "Mutations affecting amino sugar metabolism in Escherichia coli K-12." J Bacteriol 111(1);290-1. PMID: 4591481

Hopkins13: Hopkins AP, Hawkhead JA, Thomas GH (2013). "Transport and catabolism of the sialic acids N-glycolylneuraminic acid and 3-keto-3-deoxy-D-glycero-D-galactonononic acid by Escherichia coli K-12." FEMS Microbiol Lett 347(1);14-22. PMID: 23848303

Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394

Park08: Park JT, Uehara T (2008). "How bacteria consume their own exoskeletons (turnover and recycling of cell wall peptidoglycan)." Microbiol Mol Biol Rev 72(2);211-27, table of contents. PMID: 18535144

Plumbridge89: Plumbridge JA (1989). "Sequence of the nagBACD operon in Escherichia coli K12 and pattern of transcription within the nag regulon." Mol Microbiol 1989;3(4);505-15. PMID: 2668691

Plumbridge91: Plumbridge J, Kolb A (1991). "CAP and Nag repressor binding to the regulatory regions of the nagE-B and manX genes of Escherichia coli." J Mol Biol 1991;217(4);661-79. PMID: 1848637

Plumbridge91a: Plumbridge JA (1991). "Repression and induction of the nag regulon of Escherichia coli K-12: the roles of nagC and nagA in maintenance of the uninduced state." Mol Microbiol 1991;5(8);2053-62. PMID: 1766379

Rogers88: Rogers MJ, Ohgi T, Plumbridge J, Soll D (1988). "Nucleotide sequences of the Escherichia coli nagE and nagB genes: the structural genes for the N-acetylglucosamine transport protein of the bacterial phosphoenolpyruvate: sugar phosphotransferase system and for glucosamine-6-phosphate deaminase." Gene 1988;62(2);197-207. PMID: 3284790

Souza97: Souza JM, Plumbridge JA, Calcagno ML (1997). "N-acetylglucosamine-6-phosphate deacetylase from Escherichia coli: purification and molecular and kinetic characterization." Arch Biochem Biophys 1997;340(2);338-46. PMID: 9143339

UniProt12a: UniProt Consortium (2012). "UniProt version 2012-09 released on 2012-09-12 00:00:00." Database.

UniProt12b: UniProt Consortium (2012). "UniProt version 2012-11 released on 2012-11-26 00:00:00." Database.

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

UniProtGOA12: UniProt-GOA (2012). "Gene Ontology annotation based on UniPathway vocabulary mapping."

Vogler89: Vogler AP, Lengeler JW (1989). "Analysis of the nag regulon from Escherichia coli K12 and Klebsiella pneumoniae and of its regulation." Mol Gen Genet 219(1-2);97-105. PMID: 2693951

White67: White RJ, Pasternak CA (1967). "The purification and properties of N-acetylglucosamine 6-phosphate deacetylase from Escherichia coli." Biochem J 1967;105(1);121-5. PMID: 4861885

White68: White RJ (1968). "Control of amino sugar metabolism in Escherichia coli and isolation of mutants unable to degrade amino sugars." Biochem J 106(4);847-58. PMID: 4866432

Xu06d: Xu C, Hall R, Cummings J, Raushel FM (2006). "Tight binding inhibitors of N-acyl amino sugar and N-acyl amino acid deacetylases." J Am Chem Soc 128(13);4244-5. PMID: 16568996

Other References Related to Gene Regulation

Minagawa03: Minagawa S, Ogasawara H, Kato A, Yamamoto K, Eguchi Y, Oshima T, Mori H, Ishihama A, Utsumi R (2003). "Identification and molecular characterization of the Mg2+ stimulon of Escherichia coli." J Bacteriol 185(13);3696-702. PMID: 12813061

Plumbridge01: Plumbridge J (2001). "DNA binding sites for the Mlc and NagC proteins: regulation of nagE, encoding the N-acetylglucosamine-specific transporter in Escherichia coli." Nucleic Acids Res 29(2);506-14. PMID: 11139621

Plumbridge93: Plumbridge J, Kolb A (1993). "DNA loop formation between Nag repressor molecules bound to its two operator sites is necessary for repression of the nag regulon of Escherichia coli in vivo." Mol Microbiol 10(5);973-81. PMID: 7934873

Plumbridge98a: Plumbridge J, Kolb A (1998). "DNA bending and expression of the divergent nagE-B operons." Nucleic Acids Res 26(5);1254-60. PMID: 9469834


Report Errors or Provide Feedback
Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
Page generated by SRI International Pathway Tools version 18.5 on Fri Nov 28, 2014, BIOCYC13B.