A real‐time assay for monitoring Mycobacterium tuberculosis trehalose phosphate phosphatase activity

Abstract

The otsB2 gene that encodes the enzyme trehalose phosphate phosphatase (TPP) is essential for the viability of Mycobacterium tuberculosis (M. tb). TPP possesses trehalose‐6‐phosphate (T6P) phosphatase activity that is central to the production of trehalose, which acts as the primary mycolate carrier molecule during biosynthesis of the mycobacterial outer membrane (MOM). The essentiality of the otsB2 gene and the role of TPP in trehalose biosynthesis makes TPP an intriguing target for antitubercular drug development. This abstract describes an enzymatic assay that allows real‐time monitoring of the TPP activity and determination of steady‐state kinetic parameters. The assay was analyzed for reproducibility and showed a Z′ value of 0.76 when using T6P at a concentration near the determined Km. The NIH clinical collection was used as a test set to determine assay consistency under typical assay conditions. The results from these experiments gave Z‐factors ranging from 0.44–0.52. Taken together, these data show the assay to be rapid, highly reproducible, and amenable to high‐throughput screening methodologies as a basis for identifying lead compounds that inhibit TPP.