Characterization of a novel cysteine-less Cu/Zn-superoxide dismutase in Paenibacillus lautus missing a conserved disulfide bond

Abstract

Cu/Zn-superoxide dismutase (CuZnSOD) is an enzyme that binds a copper and zinc ion and also forms an intramolecular disulfide bond. Together with the copper ion as the active site, the disulfide bond is completely conserved among these proteins; indeed, the disulfide bond plays critical roles in maintaining the catalytically competent conformation of CuZnSOD. Here, we found that a CuZnSOD protein in Paenibacillus lautus (PaSOD) has no Cys residue but exhibits a significant level of enzyme activity. The crystal structure of PaSOD revealed hydrophobic and hydrogen-bonding interactions in substitution for the disulfide bond of the other CuZnSOD proteins. Also notably, we determined that PaSOD forms a homodimer through an additional domain with a novel fold at the N terminus. While the advantages of lacking Cys residues and adopting a novel dimer configuration remain obscure, PaSOD does not require a disulfide-introducing/correcting system for maturation and could also avoid misfolding caused by aberrant thiol oxidations under an oxidative environment.

Keywords: crystal structure; dimerization; disulfide; gram-positive bacteria; superoxide dismutase.

Figure 1

A three-dimensional structure of human CuZnSOD. A homodimer of human CuZnSOD (Protein Data Bank ID: 1HL5) is shown with a copper (cyan) and zinc (magenta) ion and a conserved disulfide bond (yellow). The Arg residue (colored blue) at the entry site of superoxide to the copper site is also shown. CuZnSOD, Cu/Zn-superoxide dismutase.

Figure 2

Amino acid sequence of PaSOD-1 and -2 from Paenibacillus lautus. The amino acid sequence of PaSOD-1 and -2 (without the N-terminal signal sequence) from P. lautus is aligned with that of hSOD1 and EcSodC, which was performed by ClustalW. The regions regarded as “copper amine oxidase N-terminal domain” and “superoxide dismutase [CuZnSOD] domain” are also indicated. The amino acid residues responsible for binding of a copper and zinc ion as well as the disulfide-bonding Cys residues are emphasized as indicated. EcSodC, Escherichia coli CuZnSOD; hSOD1, human CuZnSOD; PaSOD, Cys-less CuZnSOD protein in Paenibacillus lautus.

Figure 3

PaSOD-1 exhibits enzymatic activity as CuZnSOD.A, activity assay was performed using (gray) as-isolated apo form of PaSOD-1, PaSOD-1 with an equimolar amount of either (green) Zn²⁺ or (blue) Cu²⁺, and (red) PaSOD-1 with an equimolar amount of both Cu²⁺ and Zn²⁺. Following addition of an equimolar amount of both Cu²⁺ and Zn²⁺, PaSOD-1 was further treated with 1 mM EDTA, and the activity assay was performed (yellow). As a positive control, the activity of human CuZnSOD in the holo form was also assayed (black). B, size-exclusion chromatograms of PaSOD-1 proteins (20 μM in the MN buffer), which were monitored at 280 nm and normalized for comparison (left axis), were shown with the molecular mass estimated by MALS (right axis): the upper panel, PaSOD-1 (red) and PaSOD-1^CTD (blue) in the apo form: the lower panel, PaSOD-1 (red) and PaSOD-1^CTD (blue) in the holo form. CuZnSOD, Cu/Zn-superoxide dismutase; MALS, multiangle light scattering; PaSOD-1, Cys-less CuZnSOD protein in Paenibacillus lautus; PaSOD-1^CTD, C-terminal domain of PaSOD-1.

Figure 4

PaSOD-1 forms a homodimer in a novel configuration. The overall structure of PaSOD-1 is revealed by X-ray crystal structural analysis of the dataset collected at the wavelength of 1.275 Å and shown in a ribbon model using a software UCSF Chimera (Protein Data Bank ID: 8IMD). The N-terminal domain indicated as PaSOD-1^NTD is responsible for the homodimerization of PaSOD-1. PaSOD-1^CTD exhibits an immunoglobulin-fold typical of CuZnSOD (Fig. 1) and binds a copper (cyan) and zinc (magenta) ion. The ligands for binding those metal ions are shown in a stick model. PaSOD, Cys-less CuZnSOD protein in Paenibacillus lautus.

Figure 5

A novel fold of PaSOD-1^NTDplays a role in the homodimerization of PaSOD-1.A, the structure of dimeric PaSOD-1^NTD (pink and sky blue) in a ribbon model is extracted from the overall structure (Fig. 4) and rotated vertically and horizontally by 90° as indicated. β-strands and α-helix in PaSOD-1^NTD are designated as β1–β7 and α1, respectively, and valine residues forming hydrophobic clusters are shown in a stick model. B, structural comparisons between dimeric PaSOD-1^NTD and the N-terminal domain of Escherichia coli copper amine oxidase (Protein Data Bank ID: 1OAC) are performed by their alignment using Matchmaker in UCSF Chimera. PaSOD, Cys-less CuZnSOD protein in Paenibacillus lautus.

Figure 6

Hydrophobic interactions replace the conserved disulfide bond in PaSOD-1.A–C, the overall structures of (A) PaSOD-1 dimer, (B) hSOD1 (Protein Data Bank ID: 1HL5), and (C) EcSodC (Protein Data Bank ID: 1ESO) are shown in a ribbon model. The PaSOD-1 dimer is shown by aligning one of PaSOD-1^CTD (colored in sky blue) with the subunit of hSOD1 and EcSodC (also colored in sky blue). The conserved disulfide bond in hSOD1 and EcSodC is colored yellow, and the amino acid residues ligating a copper (cyan) and zinc (magenta) ion are shown in a stick model. β8 strand and loop IV in a subunit of PaSOD-1, hSOD1, and EcSodC are colored in green and white, respectively. D–F, the magnified images of the subunits of (D) PaSOD-1, (E) hSOD1, and (F) EcSodC, which are looked from the direction shown as red arrows in (A–C), are shown. G–I, the amino acid configurations between β8 strand and loop IV are schematically represented in (G) PaSOD-1, (H) hSOD1, and (I) EcSodC. EcSodC, Escherichia coli CuZnSOD; hSOD1, human CuZnSOD; PaSOD, Cys-less CuZnSOD protein in Paenibacillus lautus.

Figure 7

Disrupting the interaction between β8 and loop IV leads to decreased activity of PaSOD-1. PaSOD-1 and the proteins with indicated mutations containing an equimolar amount of a Cu²⁺ and Zn²⁺ ion (left panel) were examined with the CuZnSOD activity assay, and the activities were shown as the amount of the protein (IC₅₀) that gives 50% inhibition of the WST-1 reduction by superoxide. Besides, PaSOD-1 proteins containing Cu²⁺ and Zn²⁺ ions were incubated with 1 mM EDTA at 25 °C (middle panel) or 45 °C (right panel) for an hour, and the activity assay was then performed. Averages were shown as bars with individual data points and error bars (standard deviation). PaSOD, Cys-less CuZnSOD protein in Paenibacillus lautus.

Figure 8

Expression of endogenous PaSOD in Paenibacillus lautus NBRC 15380.A, a representative growth curve of P. lautus in 2xYT with trace elements is shown. The turbidity was monitored automatically with the rocking incubator (TVS062CA, ADVANTEC: see the Experimental procedures section) and shown as a curve in the first ∼10 h. After ∼10 h of incubation, the turbidity (the absorbance at 660 nm) exceeded 2, an upper limit for the turbidity accurately measured; therefore, the turbidity was manually measured by diluting the P. lautus cultures at the indicated time (circles). B, P. lautus was collected at the time indicated in (A) (shown as 1–11), lysed, and then analyzed by Western blotting with anti-PaSOD antibody. C, the P. lautus lysates incubated for 5 days were semipurified as described in the Experimental procedures section; the fractions eluted from the HiTrap Phenyl FF column with PBS and water were analyzed by (left) Western blotting, (middle) in-gel superoxide dismutase activity assay, and (right) immunoblotting followed by native-PAGE. The bands observed in the immunoblots (left and right) were detected with anti-PaSOD. As controls, recombinant PaSOD-1 and -2 were examined. PaSOD, Cys-less CuZnSOD protein in Paenibacillus lautus; PBS, phosphate-buffered saline.