N-acetylglucosamine-6-phosphate deacetylase

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N-acetylglucosamine-6-phosphate deacetylase in Mycobacterium smegmatis
NagA.png
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EC no. 3.5.1.25
CAS no. 9027-50-3
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In enzymology, N-acetylglucosamine-6-phosphate deacetylase (EC 3.5.1.25), also known as GlcNAc-6-phosphate deacetylase or NagA, is an enzyme that catalyzes the deacetylation of N-acetylglucosamine-6-phosphate (GlcNAc-6-P) to glucosamine-6-phosphate (GlcN-6-P):

Contents

H2O + N-acetyl-D-glucosamine 6-phosphate acetate + D-glucosamine 6-phosphate [1]
NagA Reaction NagA Reaction.png
NagA Reaction

GlcNAc-6-phosphate deacetylase is encoded by the gene NagA. [2]

This enzyme belongs to the amidohydrolase superfamily. [3] Amidohydrolases are a type of hydrolase that acts upon amide bonds. All members of the amidohydrolase family employ a TIM barrel structure, and a vast majority of members are metalloenzymes. [4] The family of enzymes is important in amino acid and nucleotide metabolism as well as biodegradation of agricultural and industrial compounds. NagA participates in amino-sugar metabolism, specifically in the biosynthesis of amino-sugar-nucleotides. [5]

Structure

NagA is a homodimeric enzyme with two domains in each dimer of the structure. [6] Each domain I comprises a (β/α)8 - barrel structural fold, also known as a TIM barrel, and contains an active site of the enzyme. Each active site consists of the catalytic site of the enzyme and the metal-binding site that are involved in substrate and metal co-factor recognition, respectively. Domain I also forms the dimeric interface with domain I of the neighboring subunit. [6] The smaller second domain of NagA enzymes comprises a β-barrel, which potentially acts to stabilize the enzyme. [6] While all members of the amidohydrolase superfamily employ a TIM-barrel structural fold, NagA in Escherichia coli (EcNagA) has a pseudo-TIM barrel enclosing the funnel-like catalytic site of the enzyme. [7] The dimer structure of NagA is considered crucial for the activity and thermostability of the enzyme. [8]

The active site of NagA in E.coli; E.coli has a mononuclear metal-binding site with a Zn ion. Active site NagA.png
The active site of NagA in E.coli; E.coli has a mononuclear metal-binding site with a Zn ion.

Metal-binding site

Amidohydrolase enzymes can bind one, two, or three metal atoms in the active site. These metals can include Zn2+, Co2+, Fe2+, Cd2+, and others. [1] EcNagA contains a mononuclear metal-binding site with a Zn2+ ion; [3] in addition, EcNagA shows a phosphate ion bound at the metal-binding site. [7] Unlike EcNagA, NagA of Mycobacterium smegmatis (MSNagA) and Bacillus subtilis (BsNagA) have binuclear metal-binding sites. MSNagA has two divalent metal ions located in each active site, which are both required for efficient catalysis and structural stability. [6] While most other bacteria species use Zn as their metal co-factor, BsNagA utilizes iron as the predominant metal in the metal-binding site. [9]

Catalytic-binding site

Most of the active site residues of EcNagA and BsNagA are conserved and share similar structural positions. A notable difference between mycobacterial NagA enzymes and NagA enzymes from other bacterial species is the presence of a cysteine at position 131. Other bacterial species have a lysine residue at this position. This cysteine is located in the flexible loop, which prevents the physiological substrate from binding. [6]

Mechanism

The catalytic mechanism for NagA enzymes proposed utilizes nucleophilic attack via a metal-coordinated water molecule or hydroxide ion. The mechanism proceeds via a strictly conserved active-site aspartic acid residue (Asp-273) that acts initially as a base to activate the hydrolytic water molecule in order to attack the carbonyl group of the substrate. [3] Asp-273 then acts as an acid to protonate the amine leaving group. One proposed mechanism using the BsNagA and its two iron co-factors in the metal-binding site demonstrates the nucleophilic attack by an Fe-bridged hydroxide and then the stabilization of the carbonyl oxygen by one of the two Fe atoms. [9]

Biological Function

NagA Pathway in Bacteria NagA Pathway in Bacteria.png
NagA Pathway in Bacteria

NagA is located in the cytoplasm of the cell. N-acetylglucosamine (GlcNAc) enters the cell as part of the breakdown of the cell wall. GlcNAc, a monosaccharide and derivative of glucose, is part of a biopolymer in the bacterial cell wall. This biopolymer forms a layered structure called peptidoglycan (PG). GlcNAc is then converted into GlcNAc-6-P by the enzyme NagE. [10] This substrate is then deacetylated into acetate and GlcN-6-P by NagA. [11] NagA is important for the production of GlcN-6-P, which is then used in two main pathways: PG recycling pathway and the glycolysis pathway.

PG recycling pathway

In the PG Recycling pathway, once GlcNAc-6-P is metabolized by NagA, its product, GlcN-6-P, can then be converted to GlcN-1-P by the enzyme GlmM, followed by reacetylation and reaction with UTP by GlmU to form UDP-GlcNAc. [10] [11] UDP-GlcNAc is the end product of this pathway, which is then used to make glycosaminoglycans, proteoglycans, and glycolipids, which are all necessary in order to replenish PG for the cell wall. [12] PG recycling is necessary for bacterial cells in order to ensure bacteria growth and prevent cell lysis. [13]

Glycolysis pathway

Instead of entering the PG recycling pathway, GlcN-6-P can be converted into fructose-6-phosphate by NagB. This reaction is reversible by the enzyme GlmS, [10] [11] an amidotransferase. [13] The produced fructose-6-phosphate then enters the glycolysis pathway. Glycolysis catalyzes the production of pyruvate, leading to the citric acid cycle and allowing for the production of amino acids. [14] GlcN-6-P and fructose-6-phosphate act as allosteric regulators of NagA, inhibiting further deacetylation of GlcNAc-6-P. [15]

Disease relevance

NagA is a potential drug target of Mycobacterium tuberculosis (Mtb). Eliminating NagA produces high levels of the allosteric activator GlcNAc-6-P, [2] which prevents the production of GlcN-6-P in order to proceed with the PG recycling pathway. NagA is, therefore, at a crucial metabolic chokepoint in Mtb, [16] representing the key enzymatic step in the generation of essential amino-sugar precursors. These precursors are required for Mtb cell wall biosynthesis and influence the PG recycling pathway. Additionally, the presence of cysteine in MSNagA's active site may represent a unique exploitative target in Mtb therapeutics. [6]

Structural studies

As of early 2019, 11 structures have been solved for this class of enzymes, with PDB accession codes 1O12, 1UN7, 1YMY, 1YRR, 2P50, 2P53, 6FV3, 6FV4, 3EGJ, 3IV8, and 2VHL.

Nomenclature

The systematic name of this enzyme class is N-acetyl-D-glucosamine-6-phosphate amidohydrolase. Other names in common use include acetylglucosamine phosphate deacetylase, acetylaminodeoxyglucosephosphate acetylhydrolase, and 2-acetamido-2-deoxy-D-glucose-6-phosphate amidohydrolase. [15]

Related Research Articles

Peptidoglycan or murein is a unique large macromolecule, a polysaccharide, consisting of sugars and amino acids that forms a mesh-like peptidoglycan layer (sacculus) that surrounds the bacterial cytoplasmic membrane. The sugar component consists of alternating residues of β-(1,4) linked N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). Attached to the N-acetylmuramic acid is an oligopeptide chain made of three to five amino acids. The peptide chain can be cross-linked to the peptide chain of another strand forming the 3D mesh-like layer. Peptidoglycan serves a structural role in the bacterial cell wall, giving structural strength, as well as counteracting the osmotic pressure of the cytoplasm. This repetitive linking results in a dense peptidoglycan layer which is critical for maintaining cell form and withstanding high osmotic pressures, and it is regularly replaced by peptidoglycan production. Peptidoglycan hydrolysis and synthesis are two processes that must occur in order for cells to grow and multiply, a technique carried out in three stages: clipping of current material, insertion of new material, and re-crosslinking of existing material to new material.

<span class="mw-page-title-main">Teichoic acid</span>

Teichoic acids are bacterial copolymers of glycerol phosphate or ribitol phosphate and carbohydrates linked via phosphodiester bonds.

N-acetylglucosamine-1-phosphate transferase is a transferase enzyme.

Uridine diphosphate <i>N</i>-acetylglucosamine Chemical compound

Uridine diphosphate N-acetylglucosamine or UDP-GlcNAc is a nucleotide sugar and a coenzyme in metabolism. It is used by glycosyltransferases to transfer N-acetylglucosamine residues to substrates. D-Glucosamine is made naturally in the form of glucosamine-6-phosphate, and is the biochemical precursor of all nitrogen-containing sugars. To be specific, glucosamine-6-phosphate is synthesized from fructose 6-phosphate and glutamine as the first step of the hexosamine biosynthesis pathway. The end-product of this pathway is UDP-GlcNAc, which is then used for making glycosaminoglycans, proteoglycans, and glycolipids.

<span class="mw-page-title-main">UDP-glucose 4-epimerase</span> Class of enzymes

The enzyme UDP-glucose 4-epimerase, also known as UDP-galactose 4-epimerase or GALE, is a homodimeric epimerase found in bacterial, fungal, plant, and mammalian cells. This enzyme performs the final step in the Leloir pathway of galactose metabolism, catalyzing the reversible conversion of UDP-galactose to UDP-glucose. GALE tightly binds nicotinamide adenine dinucleotide (NAD+), a co-factor required for catalytic activity.

<span class="mw-page-title-main">UDP-N-acetylglucosamine 2-epimerase</span> Class of enzymes

In enzymology, an UDP-N-acetylglucosamine 2-epimerase is an enzyme that catalyzes the chemical reaction

In enzymology, a glucosamine-1-phosphate N-acetyltransferase is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">Glucosamine-phosphate N-acetyltransferase</span>

In enzymology, glucosamine-phosphate N-acetyltransferase (GNA) is an enzyme that catalyzes the transfer of an acetyl group from acetyl-CoA to the primary amine in glucosamide-6-phosphate, generating a free CoA and N-acetyl-D-glucosamine-6-phosphate.

In enzymology, an UDP-N-acetylglucosamine—dolichyl-phosphate N-acetylglucosaminephosphotransferase is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">UDP-N-acetylglucosamine diphosphorylase</span> Class of enzymes

In enzymology, an UDP-N-acetylglucosamine diphosphorylase is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">DPAGT1</span> Protein-coding gene in the species Homo sapiens

UDP-N-acetylglucosamine—dolichyl-phosphate N-acetylglucosaminephosphotransferase is an enzyme that in humans is encoded by the DPAGT1 gene.

N-acetyl-D-glucosamine kinase is an enzyme that in humans is encoded by the NAGK gene.

High-mannose-oligosaccharide beta-1,4-N-acetylglucosaminyltransferase, uridine diphosphoacetylglucosamine-oligosaccharide acetylglucosaminyltransferase, acetylglucosamine-oligosaccharide acetylglucosaminyltransferase, UDP-GlcNAc:oligosaccharide beta-N-acetylglucosaminyltransferase, UDP-N-acetyl-D-glucosamine:high-mannose-oligosaccharide beta-1,4-N-acetylglucosaminyltransferase) is an enzyme with systematic name UDP-N-acetyl-D-glucosamine:high-mannose-oligosaccharide 4-beta-N-acetylglucosaminyltransferase. This enzyme catalyses the following chemical reaction

Protein <i>O</i>-GlcNAc transferase Protein-coding gene in the species Homo sapiens

Protein O-GlcNAc transferase also known as OGT or O-linked N-acetylglucosaminyltransferase is an enzyme that in humans is encoded by the OGT gene. OGT catalyzes the addition of the O-GlcNAc post-translational modification to proteins.

N,N'-diacetylchitobiose phosphorylase is an enzyme with the systematic name N,N'-diacetylchitobiose:phosphate N-acetyl-D-glucosaminyltransferase. This enzyme was found in the genus Vibrio initially but has now been found to be taken up by Escherichia coli as well as many other bacteria. One study shows that Escherichia coli can replicate on a medium that is just composed of GlcNAc a product of phosphorylation of N,N'-diacetylchitobiose as the sole source of carbon. Because E. coli can go on this medium, the enzyme is present. The enzyme has also been found in multiple eukaryotic cells as well, especially in eukaryotes that make chitin and break chitin down. It is believed that N,N'-diacetylchitobiose phosphorylase is an integral part of the phosphoenolpyruvate:glucose phosphotransferase system (PTS). It is assumed that it is involved with Enzyme Complex II of the PTS and is involved with the synthesis of chitin. The enzyme is specific for N,N'-diacetylchitobiose.

UDP-N-acetylglucosamine—undecaprenyl-phosphate N-acetylglucosaminephosphotransferase is an enzyme with systematic name UDP-N-acetyl-alpha-D-glucosamine:ditrans,octacis-undecaprenyl phosphate N-acetyl-alpha-D-glucosaminephosphotransferase. This enzyme catalyses the following chemical reaction

UDP-N-acetylglucosamine---decaprenyl-phosphate N-acetylglucosaminephosphotransferase is an enzyme with systematic name UDP-N-acetyl-alpha-D-glucosamine:trans,octacis-decaprenyl-phosphate N-acetylglucosaminephosphotransferase. This enzyme catalyses the following chemical reaction

Peptidoglycan-N-acetylglucosamine deacetylase (EC 3.5.1.104, HP310, PgdA, SpPgdA, BC1960, peptidoglycan deacetylase, N-acetylglucosamine deacetylase, peptidoglycan GlcNAc deacetylase, peptidoglycan N-acetylglucosamine deacetylase, PG N-deacetylase) is an enzyme with systematic name peptidoglycan-N-acetylglucosamine amidohydrolase. This enzyme catalyses the following chemical reaction

UDP-3-O-acyl-N-acetylglucosamine deacetylase (EC 3.5.1.108, LpxC protein, LpxC enzyme, LpxC deacetylase, deacetylase LpxC, UDP-3-O-acyl-GlcNAc deacetylase, UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase, UDP-(3-O-acyl)-N-acetylglucosamine deacetylase, UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase, UDP-(3-O-(R-3-hydroxymyristoyl))-N-acetylglucosamine deacetylase) is an enzyme with systematic name UDP-3-O-((3R)-3-hydroxymyristoyl)-N-acetylglucosamine amidohydrolase. This enzyme catalyses the following chemical reaction

N-acetylmuramic acid 6-phosphate etherase (EC 4.2.1.126, MurNAc-6-P etherase, MurQ) is an enzyme with systematic name (R)-lactate hydro-lyase (adding N-acetyl-D-glucosamine 6-phosphate; N-acetylmuramate 6-phosphate-forming). This enzyme catalyses the following chemical reaction

References

  1. 1 2 "nagA - N-acetylglucosamine-6-phosphate deacetylase - Escherichia coli (strain K12) - nagA gene & protein". www.uniprot.org. Retrieved 2019-03-14.
  2. 1 2 Alvarez-Añorve LI, Bustos-Jaimes I, Calcagno ML, Plumbridge J (October 2009). "Allosteric regulation of glucosamine-6-phosphate deaminase (NagB) and growth of Escherichia coli on glucosamine". Journal of Bacteriology. 191 (20): 6401–7. doi:10.1128/JB.00633-09. PMC   2753035 . PMID   19700525.
  3. 1 2 3 Hall RS, Xiang DF, Xu C, Raushel FM (July 2007). "N-Acetyl-D-glucosamine-6-phosphate deacetylase: substrate activation via a single divalent metal ion". Biochemistry. 46 (27): 7942–52. doi:10.1021/bi700543x. PMC   2533526 . PMID   17567047.
  4. ↑ Liu A, Huo L (2014-08-15), John Wiley & Sons Ltd (ed.), "Amidohydrolase Superfamily", eLS, John Wiley & Sons, Ltd, doi:10.1002/9780470015902.a0020546.pub2, ISBN   9780470015902
  5. ↑ Yadav V, Panilaitis B, Shi H, Numuta K, Lee K, Kaplan DL (2011-06-02). "N-acetylglucosamine 6-phosphate deacetylase (nagA) is required for N-acetyl glucosamine assimilation in Gluconacetobacter xylinus". PLOS ONE. 6 (6): e18099. Bibcode:2011PLoSO...618099Y. doi: 10.1371/journal.pone.0018099 . PMC   3107205 . PMID   21655093.
  6. 1 2 3 4 5 6 Ahangar MS, Furze CM, Guy CS, Cooper C, Maskew KS, Graham B, Cameron AD, Fullam E (June 2018). "Mycobacterium tuberculosis N-acetylglucosamine-6-phosphate deacetylase (NagA)". The Journal of Biological Chemistry. 293 (25): 9770–9783. doi: 10.1074/jbc.RA118.002597 . PMC   6016474 . PMID   29728457.
  7. 1 2 Ferreira FM, Mendoza-Hernandez G, Castañeda-Bueno M, Aparicio R, Fischer H, Calcagno ML, Oliva G (June 2006). "Structural analysis of N-acetylglucosamine-6-phosphate deacetylase apoenzyme from Escherichia coli". Journal of Molecular Biology. 359 (2): 308–21. doi:10.1016/j.jmb.2006.03.024. PMID   16630633.
  8. ↑ Mine S, Kado Y, Watanabe M, Fukuda Y, Abe Y, Ueda T, Kawarabayasi Y, Inoue T, Ishikawa K (November 2014). "The structure of hyperthermophilic β-N-acetylglucosaminidase reveals a novel dimer architecture associated with the active site". The FEBS Journal. 281 (22): 5092–103. doi: 10.1111/febs.13049 . PMID   25227262. S2CID   21178562.
  9. 1 2 Vincent F, Yates D, Garman E, Davies GJ, Brannigan JA (January 2004). "The three-dimensional structure of the N-acetylglucosamine-6-phosphate deacetylase, NagA, from Bacillus subtilis: a member of the urease superfamily". The Journal of Biological Chemistry. 279 (4): 2809–16. doi: 10.1074/jbc.M310165200 . PMID   14557261.
  10. 1 2 3 Park JT, Uehara T (June 2008). "How bacteria consume their own exoskeletons (turnover and recycling of cell wall peptidoglycan)". Microbiology and Molecular Biology Reviews. 72 (2): 211–27, table of contents. doi:10.1128/MMBR.00027-07. PMC   2415748 . PMID   18535144.
  11. 1 2 3 Plumbridge J (September 2009). "An alternative route for recycling of N-acetylglucosamine from peptidoglycan involves the N-acetylglucosamine phosphotransferase system in Escherichia coli". Journal of Bacteriology. 191 (18): 5641–7. doi:10.1128/JB.00448-09. PMC   2737974 . PMID   19617367.
  12. ↑ Milewski S, Gabriel I, Olchowy J (January 2006). "Enzymes of UDP-GlcNAc biosynthesis in yeast". Yeast. 23 (1): 1–14. doi: 10.1002/yea.1337 . PMID   16408321. S2CID   39940329.
  13. 1 2 Dhar S, Kumari H, Balasubramanian D, Mathee K (January 2018). "Cell-wall recycling and synthesis in Escherichia coli and Pseudomonas aeruginosa - their role in the development of resistance". Journal of Medical Microbiology. 67 (1): 1–21. doi: 10.1099/jmm.0.000636 . PMID   29185941.
  14. ↑ Stryer L, Tymoczko JL, Berg JM (2002). "The Citric Acid Cycle". Biochemistry. 5th Edition.
  15. 1 2 White RJ, Pasternak CA (October 1967). "The purification and properties of N-acetylglucosamine 6-phosphate deacetylase from Escherichia coli". The Biochemical Journal. 105 (1): 121–5. doi:10.1042/bj1050121. PMC   1198282 . PMID   4861885.
  16. ↑ "Molecular insights of NagA enzyme could help combat TB". News-Medical.net. 2018-07-12. Retrieved 2019-03-11.

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