Evaluation and Comparison of vs Mammalian 6-O-Phospho-Kinases: Substrate Specificity and Applications.

Sensing of peptidoglycan fragments is essential for inducing downstream signaling in both mammalian and fungal systems. The hexokinases NagK and Hxk1 are crucial enzymes for the phosphorylation of peptidoglycan molecules in order to activate specific cellular responses; however, biochemical characterization of their enzymatic specificity and efficiency has yet to be investigated in depth. Here a mass spectrometry enzymatic screen was implemented to assess substrate specificity, and an ATP coupled assay provided the quantitative kinetic profiles of these two homologous, eukaryotic enzymes.

Minimalist Tetrazine -Acetyl Muramic Acid Probes for Rapid and Efficient Labeling of Commensal and Pathogenic Peptidoglycans in Living Bacterial Culture and During Macrophage Invasion.

-Acetyl muramic acid (NAM) probes containing alkyne or azide groups are commonly used to investigate aspects of cell wall synthesis because of their small size and ability to incorporate into bacterial peptidoglycan (PG). However, copper-catalyzed alkyne-azide cycloaddition (CuAAC) reactions are not compatible with live cells, and strain-promoted alkyne-azide cycloaddition (SPAAC) reaction rates are modest and, therefore, not as desirable for tracking the temporal alterations of bacterial cell growth, remodeling, and division. Alternatively, the tetrazine--cyclooctene ligation (Tz-TCO), which is the fastest known bioorthogonal reaction and not cytotoxic, allows for rapid live-cell labeling of PG at biologically relevant time scales and concentrations.

Bioorthogonal Labeling and Click-Chemistry-Based Visualization of the Tannerella forsythia Cell Wall.

The objective of this chapter is to provide a detailed protocol for the peptidoglycan (cell wall) labeling of the periodontal pathogen Tannerella forsythia and the development of a laboratory-safe Escherichia coli strain utilizing the N-acetylmuramic acid recycling enzymes AmgK, N-acetylmuramate/N-acetylglucosamine kinase, and MurU, N-acetylmuramate alpha-1-phosphate uridylyltransferase, from T. forsythia. The procedure involves bioorthogonal labeling of bacterial cells with an azido-modified analog of the amino sugar, N-acetylmuramic acid, through "click chemistry" with a fluorescent dye.

Stress-induced non-replicating Mycobacterium smegmatis incorporates exogenous fatty acids into glycopeptidolipids.

Nontuberculous mycobacteria (NTM) such as Mycobacterium smegmatis accumulate high levels of glycopeptidolipids (GPLs) on their outer surface. The biosynthesis of GPLs is critically linked to biofilm formation by NTM which also includes opportunistic pathogens such as Mycobacterium abscessus. Although GPLs have been investigated in many earlier studies, the biosynthesis of GPLs using exogenous fatty acids in M.