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. 2015 Mar 26;81(8):2781–2796. doi: 10.1128/AEM.04221-14

FIG 8.

FIG 8

CgMsrA was reduced by Mrx1 with a monothiolic mechanism. (A) Nonmycothiolated CgMsrA was unable to create any stable association with Mrx1. Nonreducing 15% SDS-PAGE shows each CgMsrA incubated with each Mrx1 in the presence of MetO. Lanes a to d, CgMsrA(C91S), CgMsrA(C91S,C204S), CgMsrA(C91S,C213S), and CgMsrA(C91S,C204S,C213S), respectively; lane e, Mrx(C15S); lanes f to i, CgMsrA(C91S), CgMsrA(C91S,C204S), CgMsrA(C91S,C213S), and CgMsrA(C91S,C204S,C213S) incubated with Mrx1(C15S) to generate mixture proteins, respectively. (B) S-Mycothiolation of CgMsrA was monitored by biotin switch assay. IAM-alkylated protein extracts of ΔmsrA(pXMJ19-msrA) and ΔmsrA ΔmshC(pXMJ19-msrA) strains were harvested before and 30 min after H2O2 stress and subjected to His·Bind Ni-NTA resin purification. The resulting His6-CgMsrA was demycothionylated using the Mrx1/MSH/MTR system, and the free protein thiol was tagged with biotin-maleimide, followed by separation on nonreducing 15% SDS-PAGE, and blotted onto nitrocellulose membranes. The membranes were reacted with HRP-conjugated streptavidin to detect S-mycothiolation of CgMsrA and incubated with the anti-His (α-His) antibody to indicate the same amount of His6-CgMsrA used for demycothionylation analysis. (C) MALDI-TOF MS-MS analysis of CgMsrA(C91S) in the presence of both MetO and MSH (top), or MSH only (bottom). An increase of 483.1 Da was observed for CgMsrA(C91S) after treatment with MetO and MSH. (D) S-Mycothiolated CgMsrA can be reactivated by Mrx1 or Mrx1(C15S) in vitro. The Mrx1 electron transfer pathway composed of Mrx1/Mrx1(C15S), MSH, Mtr, and NADPH was reconstructed with reduced or S-mycothiolated CgMsrA added as the substrate. (E) There is no formation of heterodimers between S-mycothiolated CgMsrA(C91S,C204S,C213S) and Mrx1(C15S) in vitro. S-Mycothiolated CgMsrA(C91S,C204S,C213S) was incubated with Mrx1(C15S) in the presence of MetO, followed by separation on nonreducing 15% SDS-PAGE. Lanes: 1, Mrx1(C15S); 2, S-mycothiolated CgMsrA(C91S,C204S,C213S); 3, a mixture of Mrx1(C15S) and S-mycothiolated CgMsrA(C91S,C204S,C213S).