UDP-N-acetylenolpyruvylglucosamine reductase (MurB)

 

UDP-N-acetylenolpyruvylglucosamine reductase (MurB) reduces both E and Z isomers of enolbutyryl-UDP-GlcBAc analogs of the C3 enolpyruvate substate to UDP-methyl-N-acetylmuramic acid in the presence of NADPH. The overall product of this metabolic pathway, petidoglycan, is a biopolymer unique to Gram-positive and Gram-negative bacteria for which is essential for maintaining osmotic cell wall integrity. The absence of a homologue in eukaryotic cells makes MurB an attractive target for small molecule inhibitors with the potential to have broad antibacterial activity.

The ability of MurB to catalyse the stereo-selective reduction of both E and Z isomers of the substrate is thought to result from the active site architecture restricting free rotation around the C2-C3 bond, and slowing the rate relative to reprotonation. Structural data show the functional groups thought to be involved in the hydride transfer to C3 and protonation at C2 of the enol-ether substrate are arranged anti relative to the enol-double bond. From this information, the stereochemical outcome was predicted to yield a 2R,3R-dideuterio product. This product was later identified using chemical synthetic analysis and comparative NMR.

 

Reference Protein and Structure

Sequence
P08373 UniProt (1.3.1.98) IPR003170 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1mbb - OXIDOREDUCTASE (2.3 Å) PDBe PDBsum 1mbb
Catalytic CATH Domains
3.30.465.10 CATHdb 3.90.78.10 CATHdb (see all for 1mbb)
Cofactors
Fadh2(2-) (1), Potassium(1+) (1)
Click To Show Structure

Enzyme Reaction (EC:1.3.1.98)

NADPH(4-)
CHEBI:57783ChEBI
+
hydron
CHEBI:15378ChEBI
+
UDP-N-acetyl-3-O-(1-carboxylatovinyl)-alpha-D-glucosamine(3-)
CHEBI:68483ChEBI
→
NADP(3-)
CHEBI:58349ChEBI
+
UDP-N-acetyl-alpha-D-muramate(3-)
CHEBI:70757ChEBI
Alternative enzyme names: MurB reductase, UDP-N-acetylenolpyruvoylglucosamine reductase, UDP-N-acetylglucosamine-enoylpyruvate reductase, UDP-GlcNAc-enoylpyruvate reductase, Uridine diphospho-N-acetylglucosamine-enolpyruvate reductase, Uridine diphosphoacetylpyruvoylglucosamine reductase, Uridine-5'-diphospho-N-acetyl-2-amino-2-deoxy-3-O-lactylglucose:NADP-oxidoreductase,

Enzyme Mechanism

Introduction

The mechanism proceeds as follows: NADPH binds to the enzyme and hydride transfer of the 4-pro -S hydrogen of NADPH to N5 of an enzyme-bound flavin (FAD cofactor). Release of NADP+ is followed by the binding of UDP-GlcNAc to the enzyme active site. Hydride transfer from the reduced flavin to C3 of the enolpyruvyl moiety of the UDP-sugar substrate generates a carbanion equivalent at C2, which can be stabilised by the R -carboxylate at C1 as an enol intermediate. A proton from Ser229 is then transferred to C2, giving the UDPMurNAc product.

Catalytic Residues Roles

UniProt PDB* (1mbb)
Ser229 Ser229A Acts as a general acid to quench the enol intermediate to form the acid product. The acid/base role proposed for Ser229 is unusual. The pKa of a serine residue hydroxyl group is typically in the range of 13-15, making proton donation at neutral pH energetically unfavourable. However, in this reaction where Ser229 acts as an electrophile, it is a comparatively favourable proton donor, it is located and orientated to perform this function and loss of function mutagenesis studies indicate Ser299 to be a key catalytic residue. activator, proton acceptor, proton donor
Arg159, Glu325 Arg159A, Glu325A Acts to stabilise carbanion intermediate as an enol by hydrogen-bonding. attractive charge-charge interaction, electrostatic stabiliser, increase electrophilicity
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

hydride transfer, cofactor used, native state of cofactor is not regenerated, proton transfer, native state of cofactor regenerated, intermediate formation, overall reactant used, intermediate collapse, overall product formed, native state of enzyme regenerated, inferred reaction step

References

  1. Benson TE et al. (1997), Biochemistry, 36, 796-805. Kinetic Characterization of Wild-Type and S229A Mutant MurB:  Evidence for the Role of Ser 229 as a General Acid†. DOI:10.1021/bi962220o. PMID:9020777.
  2. Nishida S et al. (2006), J Biol Chem, 281, 1714-1724. Identification and characterization of amino acid residues essential for the active site of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from Staphylococcus aureus. DOI:10.1074/jbc.M509277200. PMID:16236703.
  3. Constantine KL et al. (1997), J Mol Biol, 267, 1223-1246. Characterization of NADP+ binding to perdeuterated MurB: backbone atom NMR assignments and chemical-shift changes. DOI:10.1006/jmbi.1997.0915. PMID:9150408.
  4. Lees WJ et al. (1996), Biochemistry, 35, 1342-1351. (E)-Enolbutyryl-UDP-N-acetylglucosamine as a Mechanistic Probe of UDP-N-acetylenolpyruvylglucosamine Reductase (MurB)†,‡. DOI:10.1021/bi952287w. PMID:8634262.

Step 1. NADPH binds in close proximity to Ser229 and transfers a hydrogen to the N5 of the enzyme bound flavin cofactor. It is uncertain which residues are specifically involved in promoting the hydride transfer from the NADPH cofactor to the enzyme bound flavin. NMR analysis indicates NADPH to bind in the vicinity of Ser229, as well as several aromatic residues [PMID:9020777]. A monovalent cation is required for catalysis, although no metal is present in the crystal structure.

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Catalytic Residues Roles

Residue Roles

Chemical Components

hydride transfer, cofactor used, native state of cofactor is not regenerated

Step 2. The flavin cofactor transfers a hydride atom from N5 to the re-face of the C2-C3 enolpyruvyl group. Either Glu325 or Arg159 could act as the proton donor: both are correctly positioned and orientated within the active site, and both are required for catalysis [PMID:8634262].

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Catalytic Residues Roles

Residue Roles
Glu325A attractive charge-charge interaction, increase electrophilicity, electrostatic stabiliser
Arg159A attractive charge-charge interaction, electrostatic stabiliser, increase electrophilicity
Glu325A proton donor

Chemical Components

hydride transfer, proton transfer, native state of cofactor regenerated, intermediate formation, overall reactant used

Step 3. Glu325 acts as a general base, taking back the enol proton. This initiates the conjugate, stereo-selective proton abstraction by the enolate from Ser229. For MerB to catalyse the stereoselective reduction of both E and Z substrate isomers, the rate of rotation of the C2-C3 bond must be slow relative to reprotonation. The active site architecture ensures stereoselective discrimination in the formation of the enantio-selective product [PMID:8634262].

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Catalytic Residues Roles

Residue Roles
Glu325A activator, attractive charge-charge interaction, electrostatic stabiliser, increase basicity
Arg159A attractive charge-charge interaction, electrostatic stabiliser
Ser229A activator
Ser229A proton donor
Glu325A proton acceptor

Chemical Components

proton transfer, intermediate collapse, overall product formed

Step 4. Reprotonation of Ser229 regenerates the active site. Proton labelling studies indicate that Ser229 is exposed to solvent, and so it is inferred that the residue is regenerated by reprotonation from a solvent molecule [PMID:8634262].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser229A proton acceptor

Chemical Components

proton transfer, native state of enzyme regenerated, inferred reaction step
Download: Image, Marvin File

Step 1. NADPH binds in close proximity to Ser229 and transfers a hydrogen to the N5 of the enzyme bound flavin cofactor. It is uncertain which residues are specifically involved in promoting the hydride transfer from the NADPH cofactor to the enzyme bound flavin. NMR analysis indicates NADPH to bind in the vicinity of Ser229, as well as several aromatic residues [PMID:9020777]. A monovalent cation is required for catalysis, although no metal is present in the crystal structure.

Step 2. The flavin cofactor transfers a hydride atom from N5 to the re-face of the C2-C3 enolpyruvyl group. Either Glu325 or Arg159 could act as the proton donor: both are correctly positioned and orientated within the active site, and both are required for catalysis [PMID:8634262].

Step 3. Glu325 acts as a general base, taking back the enol proton. This initiates the conjugate, stereo-selective proton abstraction by the enolate from Ser229. For MerB to catalyse the stereoselective reduction of both E and Z substrate isomers, the rate of rotation of the C2-C3 bond must be slow relative to reprotonation. The active site architecture ensures stereoselective discrimination in the formation of the enantio-selective product [PMID:8634262].

Step 4. Reprotonation of Ser229 regenerates the active site. Proton labelling studies indicate that Ser229 is exposed to solvent, and so it is inferred that the residue is regenerated by reprotonation from a solvent molecule [PMID:8634262].

Products.

Contributors

Sophie T. Williams, Anna Waters, Craig Porter, Gemma L. Holliday, Charity Hornby