Colorimetric Enzyme Amplification: In Vitro Analysis of Fatty Acid Synthesis and Tissue Transglutaminase Systems

One of the greatest challenges in human health is the management of multidrug resistance in bacteria responsible for infections such as pneumonia and tuberculosis. Inhibiting the type II bacterial fatty acid biosynthesis pathway (FAS-II) is a promising strategy towards circumventing drug resistance mechanisms due to its essential role in bacterial growth and membrane construction. However, an accurate method of measuring different rates of fatty acid synthesis is needed in order to truly evaluate the viability of the FAS pathway as a drug target. This project describes the use of using a novel FAS assay to quantify the rate of fatty acid synthesis by the detection of NADP⁺, a key fatty acid synthesis product. By using enzyme amplification methods to generate a colored end product from NADP⁺, the rate of NADP⁺ production can be determined under a wide variety of reaction conditions. Here we report the novel FAS assay’s use to analyze the kinetic activity in an in vitro human tissue transglutaminase (TG2) system, which produces NADP⁺ using similar processes. The change in absorbance over time was roughly proportional to the TG2 reaction rate. Further applications of the enzyme amplification assay are demonstrated by blinded experiments involving four TG2 inhibitors, which have similar inhibitory mechanisms to those inherent in many FAS inhibitors. Our findings provide a new foundation for assessing the scope and limitations of inhibiting bacterial fatty acid biosynthesis as a valid antibacterial strategy for treating drug-resistant bacteria.