The first structure of a cold-adapted superoxide dismutase (SOD): biochemical and structural characterization of iron SOD from Aliivibrio salmonicida

Abstract

Superoxide dismutases (SODs) are metalloenzymes that catalyse the dismutation of the superoxide radical anion into O(2) and H(2)O(2) in a two-step reaction. The crystal structure of the iron superoxide dismutase from the cold-adapted and fish-pathogenic bacterium Aliivibrio salmonicida (asFeSOD) has been determined and refined to 1.7 A resolution. The protein has been characterized and compared with the closely related homologous iron superoxide dismutase from the mesophilic Escherichia coli (ecFeSOD) in an attempt to rationalize its environmental adaptation. ecFeSOD shares 75% identity with asFeSOD. Compared with the mesophilic FeSOD, the psychrophilic FeSOD has distinct temperature differences in residual activity and thermostability that do not seem to be related to structural differences such as intramolecular or intermolecular ion bonds, hydrogen bonds or cavity sizes. However, an increased net negative charge on the surface of asFeSOD may explain its lower thermostability compared with ecFeSOD. Activity measurements and differential scanning calorimetry measurements revealed that the psychrophilic asFeSOD had a thermostability that was significantly higher than the optimal growth temperature of the host organism.

Figure 1

Alignment of FeSODs. Full-length alignment of the FeSOD protein product from A. salmonicida (asFeSOD) with the products of other FeSOD genes from E. coli (ecFeSOD), A. fischeri (afFeSOD), V. vulnificus (vvFeSOD), V. parahaemolyticus (vpFeSOD) and V. cholerae (vcFeSOD). Metal-coordinating residues are marked with a green triangle. Secondary-structure elements from the crystal structure of asFeSOD are indicated above the alignment.

Figure 2

SDS–PAGE of purified asFeSOD. From the left, approximately 5, 10 and 490 µg purified asFeSOD. Mark12 Unstained Standard (Invitrogen) is shown on the right. Purified asFeSOD has a monomer size of 21.4 kDa.

Figure 3

Activity and stability measurements for asFeSOD and ecFeSOD. (a) Enzymes were incubated for 30 min at temperatures ranging from 273 to 328 K for asFeSOD and from 273 to 346 K for ecFeSOD before residual activity was measured by a SOD–WST assay. The highest residual activity measured was set to 100%. (b) Thermal unfolding of asFeSOD and ecFeSOD measured by DSC at a scan rate of 1 K min⁻¹ (1 kcal = 4.184 kJ). The thermograms are baseline-subtracted and normalized for protein concentration.

Figure 4

Structural features of asFeSOD. (a) Cartoon representation of the crystallized dimer of FeSOD from A. salmonicida (asFeSOD). α-Helices are coloured red, β-strands are coloured blue and loop regions are coloured green. Fe atoms are indicated as orange spheres. (b) Close-up view of the active site of asFeSOD, illustrating the Fe coordination. The Fe atom is shown as an orange sphere, the coordinated water molecule as a red sphere and the protein ligands are shown in ball-and-stick representation. Secondary-structure elements are coloured as in (a). (c) Slabbed view of the molecular surface of the dimer of asFeSOD, illustrating the tunnel that runs through the dimer interface and connects the two active sites. The monomers are shown in cartoon representation and are coloured red and green. The view on the right is rotated 90° around x compared with that on the left.