Crystallization and preliminary X‐ray analysis of the monomeric Cu, Zn superoxide dismutase from Escherichia coli

Abstract

Abstract:The Cu,Zn superoxide dismutase (Cu,Zn SOD) originally isolated from the periplasmic space of Escherichia coli has been cloned and overexpressed in the E. coli strain BMH 71/18. The protein has been purified as a single component of 17,000 Da, corresponding to one subunit of the common dimeric eukaryotic Cu,Zn SODs. Large crystals of the purified protein have been grown in the presence of polyethylene glycol 4,000 at pH 8.5; the crystals belong to the monoclinic space group P2,, with unit cell constants a = 33.1 A, b = 52.6 A, c = 43.3 A, P = I 11.4 degrees.One SOD subunit is contained in the asymmetric unit, yielding a V,,, value of 2.1 A3/Da; the crystals diffract X-rays beyond 2.0 A resolution.Keywords: crystals; enzyme structure; Escherichia coli; metal proteins; monomer-dimer equilibrium Cu,Zn superoxide dismutases (SODs) are a class of metalloenzymes involved in the physiologic response to oxygen toxicity by their ability to catalyze the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide (Bannister et al., 1987). All the eukaryotic Cu,Zn SODs so far characterized are homodimeric enzymes containing one atom of copper and one atom of zinc per subunit. The catalytic mechanism entails alternate reduction and reoxidation of the Cu2+ ion, during successive encounters with the substrate, in a process that is thought to be dominated by an evolutionarily conserved distribution of electrostatic charges around the active site (Getzoff et al., 1983; Desideri et al., 1992). Analysis of the known crystal structure of oxidized Cu,Zn SODs from ox (Tainer et al., 1982), spinach (Kitagawaet al., 1991), yeast (Djinovic et al., 1992a), human (Parge et al. 1992), and Xenopus luevis (DjinovicReprint requests to: Alessandro Desideri, Department of Biology, University of Rome "Tor Vergata," Via della Ricerca Scientifica, 0 0 1 33 Rome, Italy; e-mail: desideri@utovrm.it. Carugo et al., 1996), as well as of the semisynthetic Cu,Co bovine SOD (Djinovic et al., 1992b) and the reduced SOD from the same source (Rypnieswski et al., 1995), have demonstrated that all these eukaryotic isoenzymes possess a nearly superimposible threedimensional fold, based on a flattened Greek-key eight-stranded P-barrel, with only minor local differences (Bordo et al., 1994). Such an exceptionally high degree of structural conservation is highlighted by the discovery (Deng et al., 1993; Rosen et al., 1993) that different point-mutations potentially able to influence the subunit fold, the dimeric association, or the active site structure of human Cu,Zn SOD are associated with the neurologic degeneration observed in familial amyotrophic lateral sclerosis (FALS).Cu,Zn SODs have also been isolated from the periplasmic space of some bacterial species. The primary sequences of the bacterial Cu,Zn SODs display a variable pattern of insertions and deletions in the loops connecting the P-strands that are expected to result in a modified subunit interface and in an altered organization of the active site channel with respect to the...

Eukaryotic copper-only superoxide dismutases (SODs): A new class of SOD enzymes and SOD-like protein domains

“…Cu/Zn-SODs can be homodimeric (SOD1), tetrameric (ecSOD), or in rare cases monomeric (Escherichia coli SodC) (3,45). Each monomer has several landmark features: a Greek key β-barrel fold, highly conserved Cu and Zn binding residues, a conserved disulfide, active site arginine, and an extended loop VII, also known in eukaryotic Cu/Zn-SODs as the electrostatic loop (ESL) (46).…”

“…Even so, within the eubacterial kingdom, Cu-only SOD enzymes appear unique to the Mycobacterium genus, as all other periplasmic SODs characterized to date have both Cu and Zn (9,34,45,57).…”

The solution structure of reduced dimeric copper zinc superoxide dismutase. The structural effects of dimerization

“…Copper occurs in the oxidized and in the reduced state, both of which are necessary for the function. The X-ray structure of the oxidized form has been available since 1982 for the bovine enzyme [6,7] and several other structures have become available [8][9][10][11][12][13][14][15][16][17][18]. Reduced state structures are also available although the picture is less clear-cut around the copper-binding site [19][20][21].…”

The solution structure of reduced dimeric copper zinc superoxide dismutase

“…Copper occurs in the oxidized and in the reduced state, both of which are necessary for the function. The X‐ray structure of the oxidized form has been available since 1982 for the bovine enzyme [6,7] and several other structures have become available [8–18]. Reduced state structures are also available although the picture is less clear‐cut around the copper‐binding site [19–21].…”