Essential Role of the Linker Region in the Higher Catalytic Efficiency of a Bifunctional MsrA-MsrB Fusion Protein

Ah Reum Han, Moon Jung Kim, Geun Hee Kwak, Jonghyeon Son, Kwang Yeon Hwang, Hwa Young Kim

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Many bacteria, particularly pathogens, possess methionine sulfoxide reductase A (MsrA) and B (MsrB) as a fusion form (MsrAB). However, it is not clear why they possess a fusion MsrAB form rather than the separate enzymes that exist in most organisms. In this study, we performed biochemical and kinetic analyses of MsrAB from Treponema denticola (TdMsrAB), single-domain forms (TdMsrA and TdMsrB), and catalytic Cys mutants (TdMsrABC11S and TdMsrABC285S). We found that the catalytic efficiency of both MsrA and MsrB increased after fusion of the domains and that the linker region (iloop) that connects TdMsrA and TdMsrB is required for the higher catalytic efficiency of TdMsrAB. We also determined the crystal structure of TdMsrAB at 2.3 Å, showing that the iloop mainly interacts with TdMsrB via hydrogen bonds. Further kinetic analysis using the iloop mutants revealed that the iloop-TdMsrB interactions are critical to MsrB and MsrA activities. We also report the structure in which an oxidized form of dithiothreitol, an in vitro reductant for MsrA and MsrB, is present in the active site of TdMsrA. Collectively, the results of this study reveal an essential role of the iloop in maintaining the higher catalytic efficiency of the MsrAB fusion enzyme and provide a better understanding of why the MsrAB enzyme exists as a fused form.

Original languageEnglish
Pages (from-to)5117-5127
Number of pages11
JournalBiochemistry
Volume55
Issue number36
DOIs
Publication statusPublished - 2016 Sep 13

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Methionine Sulfoxide Reductases
Fusion reactions
Proteins
Enzymes
Kinetics
Treponema denticola
Dithiothreitol
Reducing Agents
Pathogens
Bacteria
Hydrogen bonds
Crystal structure
Hydrogen
Catalytic Domain
IgA receptor

ASJC Scopus subject areas

  • Biochemistry

Cite this

Essential Role of the Linker Region in the Higher Catalytic Efficiency of a Bifunctional MsrA-MsrB Fusion Protein. / Han, Ah Reum; Kim, Moon Jung; Kwak, Geun Hee; Son, Jonghyeon; Hwang, Kwang Yeon; Kim, Hwa Young.

In: Biochemistry, Vol. 55, No. 36, 13.09.2016, p. 5117-5127.

Research output: Contribution to journalArticle

Han, Ah Reum ; Kim, Moon Jung ; Kwak, Geun Hee ; Son, Jonghyeon ; Hwang, Kwang Yeon ; Kim, Hwa Young. / Essential Role of the Linker Region in the Higher Catalytic Efficiency of a Bifunctional MsrA-MsrB Fusion Protein. In: Biochemistry. 2016 ; Vol. 55, No. 36. pp. 5117-5127.
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AB - Many bacteria, particularly pathogens, possess methionine sulfoxide reductase A (MsrA) and B (MsrB) as a fusion form (MsrAB). However, it is not clear why they possess a fusion MsrAB form rather than the separate enzymes that exist in most organisms. In this study, we performed biochemical and kinetic analyses of MsrAB from Treponema denticola (TdMsrAB), single-domain forms (TdMsrA and TdMsrB), and catalytic Cys mutants (TdMsrABC11S and TdMsrABC285S). We found that the catalytic efficiency of both MsrA and MsrB increased after fusion of the domains and that the linker region (iloop) that connects TdMsrA and TdMsrB is required for the higher catalytic efficiency of TdMsrAB. We also determined the crystal structure of TdMsrAB at 2.3 Å, showing that the iloop mainly interacts with TdMsrB via hydrogen bonds. Further kinetic analysis using the iloop mutants revealed that the iloop-TdMsrB interactions are critical to MsrB and MsrA activities. We also report the structure in which an oxidized form of dithiothreitol, an in vitro reductant for MsrA and MsrB, is present in the active site of TdMsrA. Collectively, the results of this study reveal an essential role of the iloop in maintaining the higher catalytic efficiency of the MsrAB fusion enzyme and provide a better understanding of why the MsrAB enzyme exists as a fused form.

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