title: A homogeneous, high-throughput-compatible, fluorescence intensity-based assay for UDP-N-acetylenolpyruvylglucosamine reductase (MurB) with nanomolar product detection.
abstract: A novel assay for the NADPH-dependent bacterial enzyme UDP-N-acetylenolpyruvylglucosamine reductase (MurB) is described that has nanomolar sensitivity for product formation and is suitable for high-throughput applications. MurB catalyzes an essential cytoplasmic step in the synthesis of peptidoglycan for the bacterial cell wall, reduction of UDP-N-acetylenolpyruvylglucosamine to UDP-N-acetylmuramic acid (UNAM). Interruption of this biosynthetic pathway leads to cell death, making MurB an attractive target for antibacterial drug discovery. In the new assay, the UNAM product of the MurB reaction is ligated to L-alanine by the next enzyme in the peptidoglycan biosynthesis pathway, MurC, resulting in hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). The ADP is detected with nanomolar sensitivity by converting it to oligomeric RNA with polynucleotide phosphorylase and detecting the oligomeric RNA with a fluorescent dye. The product sensitivity of the new assay is 1000-fold greater than that of the standard assay that follows the absorbance decrease resulting from the conversion of NADPH to NADP(+). This sensitivity allows inhibitor screening to be performed at the low substrate concentrations needed to make the assay sensitive to competitive inhibition of MurB.
authors: Bioscience Department, Infection Innovative Medicines, AstraZeneca R&D Boston, Waltham, MA 02451, USA. adam.shapiro@astrazeneca.com
authors: Adam B
authors: AB
authors: Shapiro
authors: Stephania
authors: S
authors: Livchak
authors: Ning
authors: N
authors: Gao
authors: James
authors: J
authors: Whiteaker
authors: Jason
authors: J
authors: Thresher
authors: Haris
authors: H
authors: Jahić
authors: Jian
authors: J
authors: Huang
authors: Rong-Fang
authors: RF
authors: Gu
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000737:chemistry; D002237:Carbohydrate Dehydrogenases / Q000032:analysis / Q000378:metabolism*; D002473:Cell Wall; D055808:Drug Discovery / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D005453:Fluorescence; D057166:High-Throughput Screening Assays / Q000379:methods*; D010457:Peptidoglycan / Q000096:biosynthesis; D011117:Polyribonucleotide Nucleotidyltransferase / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D010457:Peptidoglycan; D000244:Adenosine Diphosphate; D002237:Carbohydrate Dehydrogenases; C023215:UDP-N-acetylmuramate dehydrogenase; D011117:Polyribonucleotide Nucleotidyltransferase