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A high-throughput, homogenous, FRET-based assay to detect membrane-bound enzyme (MraY) activity

Adam B Shapiro (1), Carl Peters (2) 1)AstraZeneca, 2) BMG LABTECH 07/2013

The bacterial enzyme N-acetylmuramoyl-pentapeptide translocase (MraY) is a potential target of antibacterial drugs. It attaches UDP-N-acetylmuramoylpentapeptide (UNAM-pp) to lipid undecaprenyl phosphate (C55P), an essential step during bacterial cell wall biosynthesis. A FRET-based approach detects substances that inhibit MraY and can be read on BMG LABTECH's PHERAstar®. The HTS-suitable assay embeds the donor fluorophore (BODIPY FL), the MraY enzyme and C55P in a micelle. Acceptor-labelled (LRPE) UNAM-pp is then transferred by MraY to the micelle-standing C55P and FRET can occur with the micelle-embedded BODIPY FL.

In presence of MraY activity, the resulting fluorescence of the acceptor depends on reaction time and acceptor concentration. The assay allowed for determination of the IC50 value of the MraY inhibitor tunicamycin A. The ratiometric analysis eliminates fluctuations between measurements and makes it a robust assay with a high Z' value.

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