bi9b01078_si_001.pdf (785.47 kB)
Initial Kinetic Characterization of Sterile Alpha and Toll/Interleukin Receptor Motif-Containing Protein 1
journal contribution
posted on 2020-02-17, 05:31 authored by Heather
S. Loring, Janneke D. Icso, Venkatesh V. Nemmara, Paul R. ThompsonSterile 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 SAM1–2TIR 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.