In vivo production of active nickel superoxide dismutase from Prochlorococcus marinus MIT9313 is dependent on its cognate peptidase

Abstract

Metal-dependent superoxide dismutases (SODs) with a specific requirement for a manganese or iron ion for catalytic activity and copper- and zinc-dependent enzymes are essential for detoxification of superoxide anion radicals. Genome sequence analyses predict the existence of a nickel-dependent enzyme (NiSOD) as the unique SOD in oxygen-evolving marine cyanobacteria. NiSOD activity was observed in Escherichia coli when sodN and sodX (encoding a putative peptidase) from Prochlorococcus marinus MIT9313 were coexpressed.

FIG. 1.

Alignment of the N-terminal parts of SodN precursors (A) and complete SodX sequences (B) from Streptomyces species (S. avermitilis [Saver], S. coelicolor [Scoel], and S. seoulensis [Sseoul]) and the marine cyanobacteria P. marinus strains MED4, MIT9313, and SS120, Synechococcus strain WH8102, T. erythraeum strain IMS101 (Ter), and C. watsonii strain WH8501 (Cwat). The arrow indicates the proteolytic cleavage site established for Streptomyces and predicted for the cyanobacterial proteins. Sequences were loaded into and analyzed with the workbench GENESOAP written by R. Cramm (23). Alignments were generated with CLUSTALW and displayed with BOXSHADE.

FIG. 2.

Constructs for expression of P. marinus MIT9313 sodN in E. coli. psodNΔ encodes a peptide lacking residues 2 to 20. See the text for details. Arrows indicate initiation, and asterisks indicate termination codons.

FIG. 3.

Activity staining and Western blot analysis of cell extracts of E. coli SG12041(pFDX500) containing psodN, psodNΔ, or psodNX. NiCl₂ was added to the growth medium to final concentrations of 500 nM, 1 μM, 5 μM, 10 μM, 100 μM, 500 μM, or 1 mM or not added. Extracts were separated by native polyacrylamide gel electrophoresis, and Western blots were developed with an anti-FLAG antibody reacting with the FLAG epitope fused to SodN. The arrow points to the position of NiSOD activity.

FIG. 4.

Restoration of oxygen tolerance in E. coli QC2375 by sodN constructs. Precultures were grown anaerobically. Ten microliters of serial 10-fold dilutions (10⁻¹ to 10⁻⁵) were spotted onto plates containing 500 μM metal salt where indicated. Plates were incubated at 37°C under air or under a dinitrogen atmosphere. N, psodN; NΔ, psodNΔ; NX, psodNX.

FIG. 5.

SOD activity in cytoplasmic fractions of E. coli QC2375 expressing sodN constructs as determined by in gel activity staining and cytochrome c reduction assays. Cells were grown in the absence (−) or presence (+) of 500 μM NiCl₂. Fifty micrograms of soluble protein was separated by native polyacrylamide gel electrophoresis. ND, below the detection limit.