Overexpression, purification, and mechanistic study of UDP-N-acetylenolpyruvylglucosamine reductase

Abstract

The recently isolated Escherichia coli murB gene (Pucci et al., 1992) has been cloned into an expression vector and the encoded UDP-N-acetylenolpyruvylglucosamine reductase (EC 1.1.1.158) was overproduced to about 10% of soluble cell protein. The encoded 38-kDa protein has been purified to near homogeneity. It was found to be a monomer and to contain stoichiometric amounts of bound FAD which is reducible in catalytic turnover. The enzyme utilizes the 4-pro-S hydrogen of NADPH to reduce the enolpyruvyl group of UDP-N-acetylglucosamine enolpyruvate to the lactyl ether in UDP-N-acetylmuramic acid. NMR analysis of products from 2H2O and 4S-[2H]NADPH incubations establishes that a hydride from NADPH via E.FADH2 is transferred to the beta-methyl of the 3-O-lactyl moiety and a proton from solvent to the alpha-carbon of the lactyl moiety of UDP-N-acetylmuramic acid. A mechanism for this unusual enolether reduction in bacterial cell wall assembly is proposed.

Cell-wall recycling and synthesis in Escherichia coli and Pseudomonas aeruginosa – their role in the development of resistance

“…UDP-GlcNAc is further converted to UDP-MurNAc pentapeptide by the activity of the Mur group of enzymes (MurA, MurB, MurC, MurD, MurE and MurF) [31,32]. To this pentapeptide, a 55-carbon aliphatic chain called undecaprenyl pyrophosphate is attached by MraY [33].…”

Crystallization and preliminary X‐ray crystallographic studies of UDP‐N‐acetylenolpyruvylglucosamine reductase

“…The recent cloning of the murb gene (Pucci et al, 1992) and the availability of the UDPGlcNAcEP substrate from work on the preceding enzyme MurZ (MurA) (Marquardt et al, 1992) allowed overexpression and purification of 100-mg quantities of MurB for biochemical studies (Benson et al, 1993). MurB purified as a monomer with a molecular weight of 35 kDa from gel filtration analysis.…”

“…A probable mechanistic pathway of transfer of the 4-pros hydride from NADPH to the UDPGlcNAcEP substrate via reduced FAD using 4-S'H-NADPH has been defined by NMR analysis of the deuterated UDPMurNAc product. The second proton necessary for reduction comes from a hypothesized solvent exchangeable basic residue determined by analysis of the deuterated UDPMurNAc after catalysis in 'H,O (Benson et al, 1993). …”

“…Presence of the substrate UDPGlcNAcEP was absolutely required to obtain crystals. UDPGlcNAcEP was synthesized enzymatically using UDP-N-acetylglucosamine, phosphoenolpyruvate, and MurZ (MurA) and purified by HPLC as described (Benson et al, 1993). Crystals also could be grown when HEPES, pH 8.0, and Tris-HC1, pH 8.0, buffers replaced the sodium cacodylate buffer.…”

Identification and Characterization of a MurA, UDP-N-Acetylglucosamine Enolpyruvyl Transferase from Cariogenic Streptococcus Mutans

“…The cell wall of bacteria is primarily composed of peptidoglycan (PG), which is a net-like and Staphylococcus aureus have been expressed and purified 9,10,11,12) . MurA is an essential enzyme in E. coli, as its deletion is lethal to the organism, and it has no mammalian homolog 13) .…”