A Fluorescence-Based Homogeneous Assay for Measuring Activity of UDP–3-O-(R-3-Hydroxymyristoyl)-N-acetylglucosamine Deacetylase

✍ Scribed by Wen Wang; Mita Maniar; Rakesh Jain; Jeff Jacobs; Joaquim Trias; Zhengyu Yuan

• Publisher: Elsevier Science
• Year: 2001
• Tongue: English
• Weight: 104 KB
• Volume: 290
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2000.4973

✦ Synopsis

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is one of the key enzymes of bacterial lipid A biosynthesis, catalyzing the removal of the N-acetyl group of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine.

The lpxC gene is essential in Gram-negative bacteria but absent from mammalian genomes, making it an attractive target for antibacterial drug discovery. Current assay methods for LpxC are not suitable for high throughput screening, since they require multiple product separation steps and the use of radioactively labeled material that is difficult to prepare. A homogenous fluorescence-based assay was developed that uses UDP-3-O-(N-hexyl-propionamide)-Nacetylglucosamine as a surrogate substrate. This surrogate can be prepared from commercially available UDP-GlcNAc by enzymatic conversion to UDP-MurNAc, which is then chemically coupled to n-hexylamine. Following the LpxC reaction, the free amine of the deacetylation product can be derivatized by fluorescamine, thus generating a fluorescent signal. This surrogate substrate has a K m of 367 M and k cat of 0.36 s ؊1 , compared to 2 M and 1.5 s ؊1 for the natural substrate. Since no separation is needed, the assay is easily adaptable to high throughput screening. IC 50 s of LpxC inhibitors determined using this assay method is similar to those measured by traditional method with the natural substrate.