Cofactors

FAD.

Reaction Mechanism

UDP-N-acetylenolpyruvylglucosamine reductase (MurB) By Reference

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.




• Summary
• Step 1
• Step 2
• Step 3
• Step 4
• Products

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

AA	Uniprot	Uniprot Resid	PDB	PDB Resid
Arg	P08373	159	1mbb	159
Ser	P08373	229	1mbb	229
Glu	P08373	325	1mbb	325

Step Components

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



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.

The products of the reaction.

View Reaction Mechanisms in M-CSA

Reaction Parameters

There are no kinetic parameters information for this Enzyme

Associated Proteins

Citations

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• One Step Forward with Dry Surface Biofilm (DSB) of Staphylococcus aureus: TMT-Based Quantitative Proteomic Analysis Reveals Proteomic Shifts between DSB and Hydrated Biofilm.
• Specific Features of the Proteomic Response of Thermophilic Bacterium Geobacillus icigianus to Terahertz Irradiation.
• Human Erysipelothrix rhusiopathiae infection via bath water - case report and genome announcement.
• Efficient extracellular production of recombinant proteins in E. coli via enhancing expression of dacA on the genome.
• In vitro and in silico assessment of probiotic and functional properties of Bacillus subtilis DE111^®.
• Ethanol tolerance assessment in recombinant E. coli of ethanol responsive genes from Lactobacillus buchneri NRRL B-30929.
• Comparative Proteomics of Outer Membrane Vesicles from Polymyxin-Susceptible and Extremely Drug-Resistant Klebsiella pneumoniae.