Insights into the role of the metal binding site in methionine‐R‐sulfoxide reductases B

Abstract

Methionine sulfoxide reductases B (MsrBs) catalyze the reduction of methionine-R-sulfoxide via a threestep chemical mechanism including a reductase step, formation of an intradisulfide bond followed by a thioredoxin recycling process. Fifty percent of the MsrBs, including the Escherichia coli enzyme, possess a metal binding site composed of two CXXC motifs of unknown function. It is located on the opposite side of the active site. The overexpressed E. coli MsrB tightly binds one atom of zinc/iron. Substitution of the cysteines of E. coli MsrB results in complete loss of bound metal and reductase activity, and leads to a low-structured conformation of the protein as shown by CD, fluorescence, and DSC experiments. Introduction of the two CXXC motifs in Neisseria meningitidis MsrB domain leads to a MsrB that tightly binds one atom of zinc/iron, shows a strongly increased thermal stability and displays a reductase activity similar to that of the wild-type but lacking thioredoxin recycling activity. These results demonstrate the stabilizing effect of the metal and the existence of a preformed metal binding site in the nonbound metal MsrB. The data also indicate that metal binding to N. meningitidis MsrB induces subtle structural modifications, which prevent formation of a competent binary complex between oxidized MsrB and reduced thioredoxin but not between reduced MsrB and substrate. The fact that the E. coli and the N. meningitidis MsrBs exhibit a similar thermal stability suggests the existence of other structural factors in the nonbound metal MsrBs that compensate the metal bound stabilizing effect.

Insights into Function, Catalytic Mechanism, and Fold Evolution of Selenoprotein Methionine Sulfoxide Reductase B1 through Structural Analysis*

“…It was also suggested that the zinc has a structural role in this protein (9,26,31). The presence of the zinc ion within MsrB1 was clearly confirmed by our NMR assignment of MsrB1.…”

“…S5). Relative to MsrB1, the crystal structures of MsrBs from N. gonorhoeae (PDB code 1L1D) (8), X. campestris (PDB code 3HCI) (36), B. pseudomallei (PDB code 3CEZ/3CXK), S. pneumoniae (PDB code 3E0M) (31), and B. subtilis (3E0O) (31), as well as the solution structure of B. subtilis MsrB (PDB code 1XM0), have a highly similar ␤-fold consisting of two ␤-sheets, one with three strands and the other with five, which are the same as ␤-sheets found in mouse MsrB1. However, the bacterial MsrBs also have several ␣-helices on the exterior part of the protein.…”

Selenium biochemistry and its role for human health

“…135 The metal is bound through four Cys residues, and has been suggested to play a structural function. 136 4.13. Selenoprotein N Se-protein N (SelN) is a ubiquitous glycoprotein highly expressed in fetal tissues, muscle, brain and lung.…”

The Anti-Oxidant Defense System of the Marine Polar Ciliate Euplotes nobilii: Characterization of the MsrB Gene Family

“…A single CXXC motif (i.e., Cys 78 -Ser-Asn-Cys 81 ) is distinctive of En-MsrB2 which, as a consequence, should be unable to bind the zinc ion. The crucial role that this ion plays in the stabilization of the MsrB molecular structure [37,38,39], thus suggests that En-MsrB2 may rely on an increased structural flexibility to interact more effectively with carrier proteins on the way to its subcellular nuclear localization. En-MsrB4 is another structurally rather eccentric MsrB protein because it includes a third CXXC motif (i.e., Cys 42 -Ile-Leu-Cys 45 ) in the amino terminal region.…”