title: Essential Role of the Linker Region in the Higher Catalytic Efficiency of a Bifunctional MsrA-MsrB Fusion Protein.
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 (TdMsrAB(C11S) and TdMsrAB(C285S)). 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.
authors.affiliation: Department of Biosystems and Biotechnology, College of Life Sciences and Biotechnology, Korea University , Seoul 02841, Republic of Korea.
authors.forename: Ah-Reum
authors.initials: AR
authors.lastname: Han
authors.affiliation: Department of Biochemistry and Molecular Biology, Yeungnam University College of Medicine , Daegu 42415, Republic of Korea.
authors.forename: Moon-Jung
authors.initials: MJ
authors.lastname: Kim
authors.affiliation: Department of Biochemistry and Molecular Biology, Yeungnam University College of Medicine , Daegu 42415, Republic of Korea.
authors.forename: Geun-Hee
authors.initials: GH
authors.lastname: Kwak
authors.affiliation: Department of Biosystems and Biotechnology, College of Life Sciences and Biotechnology, Korea University , Seoul 02841, Republic of Korea.
authors.forename: Jonghyeon
authors.initials: J
authors.lastname: Son
authors.affiliation: Department of Biosystems and Biotechnology, College of Life Sciences and Biotechnology, Korea University , Seoul 02841, Republic of Korea.
authors.forename: Kwang Yeon
authors.initials: KY
authors.lastname: Hwang
authors.affiliation: Department of Biochemistry and Molecular Biology, Yeungnam University College of Medicine , Daegu 42415, Republic of Korea.
authors.forename: Hwa-Young
authors.initials: HY
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D002942:Circular Dichroism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins