An evolutionary path to altered cofactor specificity in a metalloenzyme

Abstract

Almost half of all enzymes utilize a metal cofactor. However, the features that dictate the metal utilized by metalloenzymes are poorly understood, limiting our ability to manipulate these enzymes for industrial and health-associated applications. The ubiquitous iron/manganese superoxide dismutase (SOD) family exemplifies this deficit, as the specific metal used by any family member cannot be predicted. Biochemical, structural and paramagnetic analysis of two evolutionarily related SODs with different metal specificity produced by the pathogenic bacterium Staphylococcus aureus identifies two positions that control metal specificity. These residues make no direct contacts with the metal-coordinating ligands but control the metal's redox properties, demonstrating that subtle architectural changes can dramatically alter metal utilization. Introducing these mutations into S. aureus alters the ability of the bacterium to resist superoxide stress when metal starved by the host, revealing that small changes in metal-dependent activity can drive the evolution of metalloenzymes with new cofactor specificity.

Fig. 1. Extensive structural similarity of S. aureus MnSOD and camSOD.

a Overlaid CD spectra (n = 1) of the (upper panel) manganese-loaded (MnSOD in blue; camSOD in gray) and (lower panel) iron-loaded (MnSOD in gold; camSOD in teal) isoforms, demonstrating similar secondary structure content of the two isozymes in solution. b Superimposed crystal structure cartoons of (upper panel) manganese-loaded forms and (lower panel) iron-loaded forms of each enzyme, with the proteins colored as per panel (a), and with manganese ions, iron ions, and waters shown as purple, orange, and red spheres, respectively. Analysis of all four structures show that the polypeptide backbones of all isoforms of the staphylococcal SODs are essentially identical to within the ~2 Å resolution of the structural data (Supplementary Tables 3–5). c No significant differences within the structural resolution were detectable in the metal coordination environment that could explain the disparate metal specificity of their catalysis.

Fig. 2. Mutation of two residues located close to the active site inverts specificity.

a Superimposed ribbon representation structures of the polypeptide backbones showing conserved (C) regions of the manganese-loaded forms of MnSOD (blue) and camSOD (gray), with all variable (V) residues highlighted (yellow and orange, respectively). b Analogous ribbon representation of the structure of S. aureus MnSOD, illustrating the localization of variable (cyan) and conserved (purple) regions of SOD sequence based on ConSurf analysis of 500 SOD sequences (calculated conservation scores represented by the color spectrum key). Approximate dimerization interface is indicated (gray). c Superimposed space-filling models of MnSOD and camSOD, colored as in panel (a), illustrating that >85% of sidechains that vary between the S. aureus SODs are surface-exposed (2.5 Ų surface exposure cutoff used). d Magnified view of the superimposed cartoon representations of MnSOD and camSOD structures, colored as in panel (a), showing the spatial location of the three residues targeted for mutagenesis in proximity to the manganese ion (purple).

Fig. 3. Differences in electronic structure and auto-oxidation of the SODs.

a The 94 GHz Mn(II) field-swept echo HFEPR spectra (upper panel) of each of the dithionite-reduced, manganese-loaded forms of the S. aureus wild type and variant SODs (labeled in the color key, top left), acquired at 5–6 K (n = 1). The lower panels show expanded views of the (left) lower and (right) upper field shoulders, respectively, which are indicated by arrows in the upper panel. SOD mutations gave rise to detectable differences in the enzymes’ HFEPR spectra and their ZFI (Supplementary Figs. 6–7 and Supplementary Table 6), demonstrating altered electronic structure and redox properties caused by the mutations. b UV/visible absorption spectra of the manganese-loaded forms of the (upper) wild type and (lower) double mutant variants of MnSOD (black and purple, respectively) and camSOD (gray and orange, respectively). MnSOD exhibits greater auto-oxidation at rest than camSOD, illustrated by increased absorbance of Mn(III) at ~480 nm, a trend which is reversed in the double mutant variants that also exhibited reciprocal changes in metal specificity (Table 1). All samples (800 µM) were equilibrated with ambient aerobic conditions in 100 mM phosphate buffer, pH 7.5, 100 mM KCl, 1 mM EDTA before spectra were acquired. Spectra were collected on independent biological replicates (n = 2), mathematically converted to extinction coefficient (ε) using the Beer-Lambert law, and a representative spectrum is shown for each variant.

Fig. 4. S. aureus camSOD evolved from a manganese-specific predecessor.

a Amino acid correlation (defined in the text as the AAQ group, blue, and the GGH group, magenta) and unrooted maximum likelihood phylogenetic tree based on alignment of amino acid (a.a.) sequences of 2691 Mn/Fe SOD homologs from bacteria (green circles), eukaryotes (sky blue circles), and archaea (red circles). Known metal-specificities of characterized enzymes (MnSOD = yellow, FeSOD = brown; camSOD = orange) are annotated with triangles. b Fragment of the alignment of selected SOD sequences of known metal specificity, with the metal-binding residues (red) and three residues in S. aureus camSOD (yellow) that were targeted for mutagenesis shown. Groups 1 and 2 of coevolving residues, identified using amino acid correlation analysis, were mapped onto the alignment, illustrating the GGH or AAQ motifs and associated correlated amino acids. The correlated residues, biological origin and metal specificity are indicated using the color scheme described in a, and sequence numbering is based on the S. aureus SODs. c Sub-tree extracted from the SOD tree in panel a containing all identified staphylococcal SOD sequences. Support values correspond to maximum likelihood bootstrap values from 100 rapid bootstrap replicates, with values >90 shown in green. The scale bar indicates number of substitutions per site. Macrococcus and the oxidase-positive staphylococci form an out-group to a strongly supported grouping of oxidase-negative staphylococci, including S. aureus, consistent with published Staphylococcaceae phylogenies^,,. Within the oxidase-negative staphylococci, all MnSOD (yellow) grouped together while camSOD homologs (orange) formed an out-group to the MnSODs.

Fig. 5. Evolution of camSOD coincided with expansion of virulence factor network.

a Circular genome alignment comparing 29 analyzed staphylococcal genomes, using S. aureus strain NCTC8325 as reference genome. The sodA gene (yellow), encoding MnSOD, is located in a genomic region enriched in essential genes (blue) that is highly conserved across the staphylococci, whereas the sodM gene (orange), encoding camSOD, is located in a variable genomic region enriched in virulence factors (green). b Expanded view of the local genomic context of the sodA (upper panel) and sodM (lower panel) genes. c Heatmap depicting the results from analysis of protein similarity networks, which identified protein families whose copy numbers (see color key) have been altered during evolution of the staphylococci (see phylogenetic tree in the upper panel). Protein families are represented on horizontal lines, and are grouped as Identified in all (black), Not identified in S. aureus (gray), S. aureus-specific (red), Enriched in S. aureus (orange) as described in the Methods. Genes inferred to be essential in analyzed S. aureus genomes are illustrated by blue bars. The lower panel illustrates the amino acid residues present at positions 19, 159, and 160 of each SOD isozyme present in the respective genome.

Fig. 6. Iron-dependent camSOD activity enables S. aureus to resist host stresses.

a The ΔsodM mutant strain (lacking the gene encoding camSOD-orange squares) of S. aureus shows greatly diminished growth in the presence of paraquat (PQ) relative to wild type (WT-blue circles) under manganese-depleted conditions imposed by the presence of the human protein complex calprotectin (CP)^,,. S. aureus cells expressing the Leu159Gly variant of camSOD from the native sodM locus (green triangles) also show reduced growth relative to the wild type. Note that the asterisk represents p < 0.05 via two-way ANOVA with Tukey’s post-test performed in Graphpad Prism when compared to wild-type bacteria grown in the same concentration of calprotectin. Multiple independent clones of the Leu159Gly variant (n = 8) were assayed at the same time as those of the wild type (n = 3) and the ΔsodM mutant strain (n = 3). Each data point represents an independent biological replicate. Black bars represent the mean, with error bars showing standard error of the mean (SEM). The diminished ability of the camSOD variant to use iron (Table 1) renders S. aureus less capable of overcoming the host immune response. b, c In-gel SOD activity assays using extracts (5.4 μg) prepared from b wild type S. aureus and c the strain expressing the Leu159Gly camSOD variant, cultured under identical conditions to those used in a, demonstrate that both forms of the SodM enzyme are detectable at similar levels (upper panels). Treatment of the extracts (left) or recombinant enzymes (right) with H₂O₂ (lower panels), a specific inhibitor of the iron-loaded form, demonstrated that the camSOD was loaded with iron in wild-type cells. The MnSOD enzyme is labeled as SodA, camSOD as SodM. This is a representative gel from a triplicate analysis of independent biological replicates (n = 3). Note that molecular weight markers were not used in these native gels, with SOD bands identified in the cell extracts by comparison of their mobility with the purified recombinant proteins. Source data for all panels are provided as a source data file.

Fig. 7. Evolution of camSOD was driven by altered metal availability in the host.

Schematic model depicting the evolution of S. aureus camSOD (orange) under selection pressure caused by exposure of the S. aureus ancestor to host-imposed manganese-starvation mediated by calprotectin. Acquisition and evolution of camSOD enabled the ancestor of S. aureus to maintain its antioxidant defense under these conditions using its iron-dependent catalysis, and coincided with its acquisition of numerous genes (green) involved in infection. Collectively, these acquired systems resulted in increased survival of S. aureus during interaction with host immune mechanisms, creating pathogenic S. aureus.