A fluorescence-based assay for measuring N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside deacetylase activity

作者：Huang Xinyi; Hernick Marcy^*

来源：Analytical Biochemistry, 2011, 414(2): 278-281.

DOI：10.1016/j.ab.2011.04.001

• MshB
• Metal-dependent deacetylase
• Metallohydrolase
• Mycothiol
• Fluorescence
• Fluorescamine

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.

• 出版日期2011-7-15
• 单位美国弗吉尼亚理工大学(Virginia Tech)