Structural characterization of a pathogenicity-related superoxide dismutase codified by a probably essential gene in Xanthomonas citri subsp. citri

Abstract

Citrus canker is a plant disease caused by the bacteria Xanthomonas citri subsp. citri that affects all domestic varieties of citrus. Some annotated genes from the X. citri subsp. citri genome are assigned to an interesting class named "pathogenicity, virulence and adaptation". Amongst these is sodM, which encodes for the gene product XcSOD, one of four superoxide dismutase homologs predicted from the genome. SODs are widespread enzymes that play roles in the oxidative stress response, catalyzing the degradation of the deleterious superoxide radical. In Xanthomonas, SOD has been associated with pathogenesis as a counter measure against the plant defense response. In this work we initially present the 1.8 Å crystal structure of XcSOD, a manganese containing superoxide dismutase from Xanthomonas citri subsp. citri. The structure bears all the hallmarks of a dimeric member of the MnSOD family, including the conserved hydrogen-bonding network residues. Despite the apparent gene redundancy, several attempts to obtain a sodM deletion mutant were unsuccessful, suggesting the encoded protein to be essential for bacterial survival. This intriguing observation led us to extend our structural studies to the remaining three SOD homologs, for which comparative models were built. The models imply that X. citri subsp. citri produces an iron-containing SOD which is unlikely to be catalytically active along with two conventional Cu,ZnSODs. Although the latter are expected to possess catalytic activity, we propose they may not be able to replace XcSOD for reasons such as distinct subcellular compartmentalization or differential gene expression in pathogenicity-inducing conditions.

Fig 1. Purification and activity of XcSOD.

(A) Size exclusion chromatography on Superdex 200 10/300 showing the native protein to be dimeric in solution, (B) SDS-PAGE indicating the purity of the final product and (C) concentration dependent enzyme activity measured as the percentage reduction in the formation of WST-1-formazan as a result of superoxide removal by XcSOD.

Fig 2. The crystal structure of XcSOD.

(A) A canonical dimeric structure compatible with the sequence signatures described in the text. Secondary structure elements are labeled and the Mn²⁺ ions are indicated as spheres. (B) The active site of XcSOD showing the ligands to the metal ion and the corresponding electron density (2F_obs-F_calc contoured at 1σ).

Fig 3. Alignment of MnSODs from Xanthomonas citri (XcSOD) and Bacillus subtilis together with the FeSOD from E. coli and Xac3.

Secondary structure elements are highlighted in red (α-helices) and purple (β-strands). The colored dots indicate metal-binding residues (grey), active site vicinal residues (yellow) and the glutamine which donates a hydrogen bond to the ligand (green). The latter resides in a different position in the sequences for Mn and Fe bearing enzymes. Residues which are characteristic of either Mn-containing enzymes or Fe-containing enzymes are boxed with green or blue backgrounds respectively, whilst those characteristic of dimers are boxed in orange. These residues have been derived from previous alignment studies [24,25] and the B. subtilis and E. coli sequences presented here are used merely as representatives of the two groups of enzyme.

Fig 4. The electrostatics of the entrance channels of MnSODs.

All electrostatic potentials have been coloured using the same scale so that they are strictly comparable. The enzymes from X. citri and B. subtilis are both dimeric whilst those from S. cerevisiae and H. sapiens are tetrameric. In the latter cases a dimer has been isolated from the tetramer for the purposes of calculating the electrostatic potential. The black oval highlights the ring of positive electrostatic potential around the active site. Although the details vary from structure to structure they all share this common feature.

Fig 5. FeSOD active sites.

(A) and (B) show the active sites of the FeSOD from E. coli and Xac3 respectively. The metal ion and its coordinated water-derived ligand are shown as orange and red spheres respectively. His31 and Tyr35 have been substituted by Cys and Glu respectively leading to dramatic changes to the chemical characteristics of the active site. These include the electrostatic potential which is more negative in the case of Xac3, disfavoring the approach of the incoming superoxide substrate (C and D).