title: A fluorescence-based assay for measuring N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside deacetylase activity.
abstract: Here we report a new fluorescence-based assay for measuring MshB (N-acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-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-α-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.
authors.affiliation: Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA.
authors.forename: Xinyi
authors.initials: X
authors.lastname: Huang
authors.forename: Marcy
authors.initials: M
authors.lastname: Hernick
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism; D000581:Amidohydrolases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry*; D007700:Kinetics; D009161:Mycobacterium / Q000201:enzymology; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D000581:Amidohydrolases; C417275:N-acetyl-1-D-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside deacetylase; D000117:Acetylglucosamine