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Identification and characterization of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from the Gram-positive pathogen Streptococcus pneumoniae.
Biochem J. 2001 Apr 15; 355(Pt 2):431-5.BJ

Abstract

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.

Authors+Show Affiliations

Department of Anti-Infective Research, SmithKline Beecham Pharmaceuticals, 1250 South Collegeville Road, Collegeville, PA 19426, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

11284731

Citation

Sylvester, D R., et al. "Identification and Characterization of UDP-N-acetylenolpyruvylglucosamine Reductase (MurB) From the Gram-positive Pathogen Streptococcus Pneumoniae." The Biochemical Journal, vol. 355, no. Pt 2, 2001, pp. 431-5.
Sylvester DR, Alvarez E, Patel A, et al. Identification and characterization of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from the Gram-positive pathogen Streptococcus pneumoniae. Biochem J. 2001;355(Pt 2):431-5.
Sylvester, D. R., Alvarez, E., Patel, A., Ratnam, K., Kallender, H., & Wallis, N. G. (2001). Identification and characterization of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from the Gram-positive pathogen Streptococcus pneumoniae. The Biochemical Journal, 355(Pt 2), 431-5.
Sylvester DR, et al. Identification and Characterization of UDP-N-acetylenolpyruvylglucosamine Reductase (MurB) From the Gram-positive Pathogen Streptococcus Pneumoniae. Biochem J. 2001 Apr 15;355(Pt 2):431-5. PubMed PMID: 11284731.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Identification and characterization of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from the Gram-positive pathogen Streptococcus pneumoniae. AU - Sylvester,D R, AU - Alvarez,E, AU - Patel,A, AU - Ratnam,K, AU - Kallender,H, AU - Wallis,N G, PY - 2001/4/4/pubmed PY - 2001/6/2/medline PY - 2001/4/4/entrez SP - 431 EP - 5 JF - The Biochemical journal JO - Biochem J VL - 355 IS - Pt 2 N2 - 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. SN - 0264-6021 UR - https://www.unboundmedicine.com/medline/citation/11284731/Identification_and_characterization_of_UDP_N_acetylenolpyruvylglucosamine_reductase__MurB__from_the_Gram_positive_pathogen_Streptococcus_pneumoniae_ L2 - https://portlandpress.com/biochemj/article-lookup/doi/10.1042/0264-6021:3550431 DB - PRIME DP - Unbound Medicine ER -