Methionine Sulfoxide Reductase A (MsrA) and Its Function in Ubiquitin-Like Protein Modification in Archaea

Abstract

Methionine sulfoxide reductase A (MsrA) is an antioxidant enzyme found in all domains of life that catalyzes the reduction of methionine-S-sulfoxide (MSO) to methionine in proteins and free amino acids. We demonstrate that archaeal MsrA has a ubiquitin-like (Ubl) protein modification activity that is distinct from its stereospecific reduction of MSO residues. MsrA catalyzes this Ubl modification activity, with the Ubl-activating E1 UbaA, in the presence of the mild oxidant dimethyl sulfoxide (DMSO) and in the absence of reductant. In contrast, the MSO reductase activity of MsrA is inhibited by DMSO and requires reductant. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis reveals that MsrA-dependent Ubl conjugates are associated with DNA replication, protein remodeling, and oxidative stress and include the Ubl-modified MsrA, Orc3 (Orc1/Cdc6), and Cdc48d (Cdc48/p97 AAA+ ATPase). Overall, we found archaeal MsrA to have opposing MSO reductase and Ubl modifying activities that are associated with oxidative stress responses and controlled by exposure to mild oxidant.

In vitro Analysis of Ubiquitin-like Protein Modification in Archaea

“…While E1 homologs are widespread in Archaea, canonical E2 or E3 enzymes are not predicted in most Archaea based on primary sequence comparisons. Our recent study of Haloferax volcanii , shows methionine sulfoxide reductase A (MsrA) is needed for Ubl protein modification (sampylation) together with UbaA under a mild oxidative condition in vivo and in vitro (Fu et al , 2017). Here, we describe a detailed in vitro protocol to reconstitute and analyze MsrA-dependent sampylation.…”

“…Haloferax volcanii LR03 ( Δsamp1 Δsamp2 Δsamp3 ΔmsrA ΔubaA ) carrying plasmid pJAM3010 (a plasmid encoding msrA-strepII under control of the P2 rrnA constitutive promoter) (Fu et al , 2017) or pJAM1209 (a plasmid encoding his 6 -ubaA under control of the P2 rrnA constitutive promoter) (Hepowit et al , 2016) or without carrying any plasmid…”

“…Escherichia coli Rosetta (DE3) (Novagen, Merck, catalog number: 70954-3) carrying plasmid pJAM3200 (a plasmid encoding msrA-strepII under control of T7 promoter) from the Maupin-Furlow lab (Fu et al , 2017) or plasmid pJAM1132 (a plasmid encoding flag-his 6 -samp2 under control of T7 promoter) from the Maupin-Furlow lab (Hepowit et al , 2016)…”

“…Terminate reactions at different time points to determine the optimal reaction time. In vitro reconstitution of MsrA-dependent sampylation has been published in mBio (Fu et al , 2017). Figure 1 for the representative data is originally published as Figure 4A in our previous paper (Fu et al , 2017).…”

“…In vitro reconstitution of MsrA-dependent sampylation has been published in mBio (Fu et al , 2017). Figure 1 for the representative data is originally published as Figure 4A in our previous paper (Fu et al , 2017). The 50 kDa band corresponds to monosampylated UbaA (Figure 1, lane 12).…”