Expression, Reconstitution, and Mutation of Recombinant Streptomyces coelicolor NiSOD

Abstract

Nickel-dependent superoxide dismutases (NiSODs) represent a novel solution to controlling the deleterious effects of reactive oxygen species derived from superoxide in biology. The expression of recombinant Streptomyces coelicolor NiSOD and its in vitro processing and reconstitution to yield fully active enzyme is reported. The results of studies of NiSODs involving mutations in two putative nickel binding ligands are also reported. These studies show that mutation of M28, a strictly conserved residue and one of only three S-donor ligands in the enzyme, has no measurable effect on the spectroscopic or catalytic properties of the enzyme. In contrast, mutation of the strictly conserved N-terminal H residue has dramatic effects on both the spectroscopic and catalytic properties. These results provide insights into structural and mechanistic aspects of the novel nickel-containing reactive site.

Solution Structure of a Functional Biomimetic and Mechanistic Implications for Nickel Superoxide Dismutases

“…[14,15] It is quite interesting that selected mutation (His1!Ala) in the enzyme not only destroyed the catalytic activity [15] but also changed the preference for the oxidation state of nickel, which in the mutant is now diamagnetic Ni II . [12] This corresponds quite well with the fact that an ESR signal for Ni III could not be detected in solutions of the biomimetic. We postulate that the trans conformation of Pro5 is responsible for this change in oxidation state preference because the central nickel ion is now forced to employ a carbonyl group as the fifth ligand.…”

“…[13,16] In accord with these findings, the crystal structures contain two different nickel species, a square planar N,N,S,S-coordination geometry for the reduced Ni II form, and a square pyramidal geometry for the Ni III form in which the imidazole ring of His1 is axially coordinated (species 1 and 3 in Scheme 1). [14,15] The current understanding of the mode of action of NiSOD is based on experimental data [11][12][13][14][15][16] and operates on the assumption of an inner-sphere mechanism, which is initiated when O 2 C À (after having entered the active site possibly through an electrostatic channel [15] ) binds to the vacant axial site of Ni II on the side opposite to the His1 residue. It is possible that Tyr9 moves aside upon approach of O 2 C À , thus providing a more direct access to the central ion.…”

“…This is in clear contrast to enzymatic mutation experiments which assign an essential function to His1. [12,15] SOD activity of the Ni…”

New Insight into the Mode of Action of Nickel Superoxide Dismutase by Investigating Metallopeptide Substrate Models

“…[2] Superoxide dismutases (SODs; EC 1.15.1.1) destroy the superoxide anion radical by converting it into hydrogen peroxide and oxygen with a rate near the diffusion limit (k cat > 2 10 9 m À1 s À1 ). [3][4][5] Initially, two independent classes of SODs were identified. They contain either a dinuclear Cu or Zn cofactor or a mononuclear Fe or Mn cofactor.…”

Oxidative Processes and Antioxidative Metaloenzymes

“…NiSOD is the most recent class of SOD, which was discovered in Streptomyces [205] and cyanobacteria [144]. On the basis of amino acid sequence, metal ligand environment, and spectroscopic properties, NiSOD is distinct from other known SODs [28,87]. However, all SODs are known to have very similar catalytic rate constants, pH dependence, and catalytic functions [200].…”