Metabolic Labeling and Visualization of Isoprenoids Using Alkyne-Modified DMAPP Analogs in Bacillus subtilis.

Area of Science:

• Microbiology
• Biochemistry
• Chemical Biology

Background:

• Isoprenoids are vital bacterial metabolites involved in respiration and cell envelope synthesis.
• Limited chemical tools exist for studying isoprenoid metabolism due to precursor polarity and impermeability.

Purpose of the Study:

• To develop cell-permeant precursors for bioorthogonal labeling of isoprenoid metabolism in Bacillus subtilis.
• To investigate the metabolic suitability and labeling efficiency of alkynylated dimethylallyl pyrophosphate (DMAPP) surrogates.

Main Methods:

• Synthesis of cell-permeant alkynylated DMAPP precursors.
• Growth rescue and toxicity assays to evaluate precursor suitability.
• Metabolic incorporation studies and copper-catalyzed alkyne-azide cycloaddition (CuAAC) for visualization and profiling.

Main Results:

• A Z-alkynyl DMAPP analog demonstrated biosynthetically productive surrogate activity.
• Regiospecific labeling of membrane isoprenoids like menaquinone-7 (MK-7) and bactoprenol was achieved.
• Labeling efficiency was influenced by competition with endogenous isopentenyl pyrophosphate (IPP) and DMAPP.

Conclusions:

• Established a general chemical platform for bioorthogonal tagging of bacterial C5 metabolism.
• Enabled direct visualization and mass-spectrometric profiling of isoprenoid localization and metabolites.