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The aconitase family of proteins includes three classes of hydro-lyase enzymes: aconitases, homoaconitases and isopropylmalate (IPM) isomerases. They have a common Fe-S cluster-binding site and catalyze the isomerization of specific substrates by sequential dehydration and hydration. The archaeon Methanococcus jannaschii contains two aconitase family proteins, IPM isomerase and homoaconitase, which have 50% sequence identity. These two enzymes are heterodimeric proteins composed of large and small subunits encoded by separate genes. Although structures have been reported for the small subunits of the two enzymes, the first structures of oxidized and reduced forms of the large subunit of IPM isomerase (ox-MJ0499 and red-MJ0499, respectively) from M. jannaschii are reported here at 1.8 and 2.7 Å resolution, respectively, together with the structure of the large subunit of homoaconitase (MJ1003) at 2.5 Å resolution. The structures of both proteins have unbound Fe-S clusters and contain a fourth cysteine in the active site. The active site of MJ1003 is homologous to that of aconitase, whereas MJ0499 has significant structural distortion at the active site compared with aconitase. In addition, significant large conformational changes were observed in the active site of red-MJ0499 when compared with ox-MJ0499. The active sites of the two proteins adopt two different states before changing to the Fe-S cluster-bound `activated' state observed in aconitase. MJ1003 has an `open' active site, which forms an active pocket for the cluster, while ox-MJ0499 has a `closed' active site, with four cysteines in disulfide bonds. These data will be helpful in understanding the biochemical mechanism of clustering of the Fe-S protein family.

Clostridium oremlandii MsrA (CoMsrA) is a natively selenocysteine-containing methionine-S-sulfoxide reductase and classified into a 1-Cys type MsrA. CoMsrA exists as a monomer in solution. Herein, we report evidence that CoMsrA can undergo homodimerization during catalysis. The monomeric CoMsrA dimerizes in the presence of its substrate methionine sulfoxide via an intermolecular disulfide bond between catalytic Cys16 residues. The dimeric CoMsrA is resolved by the reductant glutaredoxin, suggesting the relevance of dimerization in catalysis. The dimerization reaction occurs in a concentration- and time-dependent manner. In addition, the occurrence of homodimer formation in the native selenoprotein CoMsrA is confirmed. We also determine the crystal structure of the dimeric CoMsrA, having the dimer interface around the two catalytic Cys16 residues. A central cone-shaped hole is present in the surface model of dimeric structure, and the two Cys16 residues constitute the base of the hole. Collectively, our biochemical and structural analyses suggest a novel dimerization-mediated mechanism for CoMsrA catalysis that is additionally involved in CoMsrA regeneration by glutaredoxin.

We previously determined the crystal structures of 1-Cys type selenoprotein MsrA from Clostridium oremlandii (CoMsrA). The overall structure of CoMsrA is unusual, consisting of two domains, the N-terminal catalytic domain and the C-terminal distinct helical domain which is absent from other known MsrA structures. Deletion of the helical domain almost completely abolishes the catalytic activity of CoMsrA. In this study, we determined the crystal structure of the helical domain-deleted (ΔH-domain) form of CoMsrA at a resolution of 1.76 Å. The monomer structure is composed of the central rolled mixed β-sheet surrounded by α-helices. However, there are significant conformational changes in the N- and C-termini and loop regions of the ΔH-domain protein relative to the catalytic domain structure of full-length CoMsrA. The active site structure in the ΔH-domain protein completely collapses, thereby causing loss of catalytic activity of the protein. Interestingly, dimer structures are observed in the crystal formed by N-terminus swapping between two molecules. The ΔH-domain protein primarily exists as a dimer in solution, whereas the full-length CoMsrA exists as a monomer. Collectively, this study provides insight into the structural basis of the essential role of the helical domain of CoMsrA in its catalysis.