Although the characterization of genes associated with cytoplasmic male sterility (CMS) and fertility restoration (Rf) has been well documented, the evolutionary relationship between nuclear Rf and CMS factors in mitochondria in Oryza species is still less understood. Here, 41 accessions from 7 Oryza species with AA genome were employed for analyzing the evolutionary relationships between the CMS factors and Rf candidates on chromosome 10. The phylogenetic tree based on restriction fragment length polymorphism patterns of CMS-associated mitochondrial genes showed that these 41 Oryza accessions fell into 3 distinct groups. Another phylogenetic tree based on PCR profiles of the nuclear Rf candidates on chromosome 10 was also established, and three groups were distinctively grouped. The accessions in each subgroup/group of the two phylogenetic trees are well parallel to each other. Furthermore, the 41 investigated accessions were test-crossed with Honglian (gametophytic type) and Wild-abortive (sporophytic type) CMS, and 5 groups were classified according to their restoring ability. The accessions in the same subgroup of the two phylogenetic trees shared similar fertility restoring pattern. Therefore, we conclude that the CMS-associated mitotypes are compatible to the Rf candidate-related nucleotypes, CMS and Rf have a parallel evolutionary relation in the Oryza species.
The catalytic active site of Mn-specific SOD (MnSOD) is organized around a redox-active Mn ion. The most highly-conserved difference between MnSODs and the homologous FeSODs is the origin of a Gln in the second coordination sphere. In MnSODs it derives from the C-terminal domain whereas in FeSODs it derives from the N-terminal domain, yet its side chain occupies almost superimposable positions in the two types of SODs’ active sites. Mutation of this Gln69 to Glu in E. coli FeSOD increased the Fe3+/2+ reduction midpoint potential by > 0.6 V without disrupting the structure or Fe binding [E. Yikilmaz, D. W. Rodgers and A.-F. Miller (2006) Biochemistry 45(4) 1151–1161]. We now describe the analogous Q146E mutant of MnSOD, explaining its low Mn content in terms increased stability of the apo-Mn protein. In 0.8 M guanidinium HCl, the Q146E-apoMnSOD displays an apparent melting midpoint temperature (Tm) 35 °C higher that of WT-apoMnSOD, whereas the Tm of WT-holoMnSOD is only 20 °C higher than that of WT-apoMnSOD. In contrast, the Tm attributed to Q146E-holoMnSOD is 40 °C lower than that of Q146E-apoMnSOD. Thus our data refute the notion that the WT residues optimize structural stability of the protein, being instead consistent with conservation on the basis of enzyme function and therefore ability to bind metal ion. We propose that the WT-MnSOD protein conserves a destabilizing amino acid at position 146 as part of a strategy for favoring metal ion binding.
The active sites of Fe‐containing SOD and Mn‐containing SOD (FeSOD and MnSOD respectively) are organized around a single redox‐active ion, Fe or Mn, which alternates between its 3+ and 2+ states during turnover. The proteins are homologues, and some members of the family support activity with either Fe or Mn (Fe&MnSODs), while others bind either metal ion but display activity with only one. A second‐sphere Gln/His residue is the most commonly conserved difference between FeSODs and MnSODs, which supply the Gln from postion 69 or 146, respectively. Mutation of Gln69 of E. coli FeSOD to Glu or His produced large changes in the E° and catalytic activity indicating that the residue at position 69 has a large influence on the affinity for at least one of the Fe oxidation states (Yikilmaz et al 2006, Biochemistry 45:1151). Analogous studies of MnSOD have found that replacement of Gln146 results in changes in activity and redox tuning, but the Q146E‐MnSOD mutant has evaded study because it fails to bind Mn. We have compared the stability and metal ion binding of a series of mutants of the MnSOD protein wherein different amino acids occupy position 146. These studies shed light on the relationship between metal ion binding and protein stability in this series of related variants of MnSOD. In particular, Q146E‐apoMn‐SOD displays a melting temperature elevated over that of WT‐apoMn‐SOD by 30 °C, and other mutations produce intermediate behaviour. Thus the WT apoMn‐SOD is less thermally stable than many of the variants. We propose that the WT protein conserves a destabilizing amino acid at position 146 as part of a strategy for favouring metal ion binding, as well as redox tuning.
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