Overexpression, purification, and mechanistic study of UDP-N-acetylenolpyruvylglucosamine reductase.
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.
Benson
T E
TE
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Marquardt
J L
JL
Marquardt
A C
AC
Etzkorn
F A
FA
Walsh
C T
CT
D001483:Base Sequence; D002237:Carbohydrate Dehydrogenases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D005798:Genes, Bacterial; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D002237:Carbohydrate Dehydrogenases; C023215:UDP-N-acetylmuramate dehydrogenase