Initial Kinetic Characterization of Sterile Alpha and Toll/Interleukin Receptor Motif-Containing Protein 1

Abstract

Sterile alpha and toll/interleukin receptor (TIR) motif-containing protein 1 (SARM1) plays a pivotal role in triggering the neurodegenerative processes that underlie peripheral neuropathies, traumatic brain injury, and neurodegenerative diseases. Importantly, SARM1 knockdown or knockout prevents degeneration, thereby demonstrating that SARM1 is a promising therapeutic target. Recently, SARM1 was shown to promote neurodegeneration via its ability to hydrolyze NAD(+), forming nicotinamide and ADP ribose (ADPR). Herein, we describe the initial kinetic characterization of full-length SARM1, as well as the truncated constructs corresponding to the SAM(1-2)TIR and TIR domains, highlighting the distinct challenges that have complicated efforts to characterize this enzyme. Moreover, we show that bacterially expressed full-length SARM1 (kcat/KM = 6000 +/- 2000 M(-1) s(-1)) is at least as active as the TIR domain alone (kcat/KM = 1500 +/- 300 M(-1) s(-1)). Finally, we show that the SARM1 hydrolyzes NAD(+) via an ordered uni-bi reaction in which nicotinamide is released prior to ADPR.