Journal
ANALYTICAL BIOCHEMISTRY
Volume 414, Issue 2, Pages 278-281Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2011.04.001
Keywords
MshB; Metal-dependent deacetylase; Metallohydrolase; Mycothiol; Fluorescence; Fluorescamine
Funding
- Jeffress Memorial Trust [J960]
Ask authors/readers for more resources
Here we report a new fluorescence-based assay for measuring MshB (N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside deacetylase) activity. The current assay for measuring MshB activity requires the fluorescent labeling of reaction mixtures and subsequent analysis using high-performance liquid chromatography (HPLC), resulting in a significant amount of processing time per sample. Here we describe a more rapid fluorescnce-based assay for the measurement of MshB activity that does not require HPLC analysis and can be carried out in multiwell plates. This fluorescamine (FSA)-based assay was used to determine the steady-state parameters for the deacetylation of N-acetyl-glucosamine (GlcNAc) by MshB, and the results from these experiments support the hypothesis that the inositol moiety primarily contributes to the affinity of GlcNAc-Ins (N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside) for MshB. The rapid nature of this assay will aid efforts toward a more detailed biochemical characterization of MshB. Furthermore, because this assay relies on the formation of a primary amine, it could be adapted to measure the activity of mycothiol-S-conjugate amidase, a metal-dependent amidase that is a potential drug target involved in the mycothiol detoxification pathway. (C) 2011 Elsevier Inc. All rights reserved.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available