Identification and characterization of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from the Gram-positive pathogen Streptococcus pneumoniae.
The UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from a Gram-positive pathogen, Streptococcus pneumoniae, was identified and characterized. The enzyme from S. pneumoniae shows 31% identity with the MurB protein from Escherichia coli, and contains the catalytic residues, substrate-binding residues and FAD-binding motif identified previously in the E. coli protein. The gene was cloned into the pET28a+ expression vector, and the 34.5 kDa protein that it encodes was overexpressed in E. coli strain BL21(DE3) to 30% of total cell protein. The majority of the protein was found to be insoluble. A variety of methods were used to increase the amount of soluble protein to 10%. This was then purified to near homogeneity in a two-step process. The absorption spectrum of the purified protein indicated it to be a flavoprotein, like its E. coli homologue, with a characteristic absorption at 463 nm. The enzyme was shown to be active, reducing UDP-N-acetylglucosamine enolpyruvate with the concomitant oxidation of NADPH, and was characterized kinetically with respect to its two substrates. The enzyme showed properties similar to those of its E. coli counterpart, being activated by univalent cations and being subject to substrate inhibition. The characterization of an important cell wall biosynthesis enzyme from a Gram-positive pathogen provides a good starting point for the discovery of antibacterial agents against MurB.
Sylvester
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DR
Department of Anti-Infective Research, SmithKline Beecham Pharmaceuticals, 1250 South Collegeville Road, Collegeville, PA 19426, USA.
Alvarez
E
E
Patel
A
A
Ratnam
K
K
Kallender
H
H
Wallis
N G
NG
D000595:Amino Acid Sequence; D001483:Base Sequence; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D007700:Kinetics; D008969:Molecular Sequence Data; D012689:Sequence Homology, Nucleic Acid; D012995:Solubility; D013296:Streptococcus pneumoniae / Q000201:enzymology*
D017931:DNA Primers; D002237:Carbohydrate Dehydrogenases; C023215:UDP-N-acetylmuramate dehydrogenase