Examination of the nickel site structure and reaction mechanism in Streptomyces seoulensis superoxide dismutase.

Superoxide dismutases are metalloenzymes involved in protecting cells from oxidative damage arising from superoxide radical or reactive oxygen species produced from superoxide. Examples of enzymes containing Cu, Mn, and Fe as the redox-active metal have been characterized. Recently, a SOD containing one Ni atom per subunit was reported. The amino acid sequence of the NiSOD deduced from the nucleotide sequence of the structural gene sodN from Streptomyces seoulensis is reported and has no homology with other SODs. X-ray absorption spectroscopic studies coupled with EPR of the Ni center show that the Ni in the oxidized (as isolated) enzyme is in a five-coordinate site composed of three S-donor ligands, one N-donor, and one other O- or N-donor. This unique coordination environment is modified by the loss of one N- (or O-) donor ligand in the dithionite-reduced enzyme. The NiSOD activity was determined by pulse radiolysis, and a value of kcat = 1.3 x 10(9) M-1 s-1 per Ni was obtained. The rate is pH sensitive and drops off rapidly above pH 8. The results characterize a novel class of metal center active in catalyzing the redox chemistry of superoxide and, when placed in context with other nickel enzymes, suggest that thiolate ligation is a prerequisite for redox-active nickel sites in metalloenzymes.

Streptomyces seoulensis sp. nov.

The taxonomic position of an actinomycete strain isolated from Korean soil was examined by a polyphasic approach. The isolate, designated IMSNU-1, was clearly assigned to the genus Streptomyces on the basis of morphological and chemotaxonomic data. The test strain was the subject of a probabilistic identification study using the identification matrices generated by Langham et al. (J. Gen. Microbiol. 135:121-133, 1989) and found to be marginally close to clusters 19 and 39. An almost complete 16S rRNA gene (rDNA) sequence was obtained for the test strain and compared with those of representative streptomycetes. 16S rDNA sequence data not only support the strain's membership in the genus Streptomyces but also provide strong evidence that our isolate is genealogically distant from representatives of clusters 19 and 39, forming a separate phyletic line in a clade encompassed by streptomycetes. It is therefore proposed from the polyphasic evidence that strain IMSNU-1 be classified in the genus Streptomyces as Streptomyces seoulensis sp. nov.

Unique isozymes of superoxide dismutase in Streptomyces griseus.

Two unique isozymes of superoxide dismutase (EC 1.15.1.1) were purified to apparent homogeneity from Streptomyces griseus by a purification procedure consisting of ammonium sulfate precipitation and chromatographies on DEAE Sephacel, Sephacryl S-200, and DEAE 5PW. Superoxide dismutase I was composed of four identical subunits of 13.0 kDa. The absorption spectrum of superoxide dismutase I exhibited absorption bands at 276 and 378 nm and a broad shoulder at 530 nm. The g values of electron paramagnetic resonance spectrum of superoxide dismutase I were g1 = 2.304, g2 = 2.248, and g3 = 2.012 and the resonance centered at g3 = 2.012 was split into triplet, indicating nickel-containing superoxide dismutase. Superoxide dismutase I contained 0.89 g-atom of nickel per mole of 13.0-kDa subunit. Superoxide dismutase II was composed of four identical subunits of 22.0 kDa. The absorption spectrum of superoxide dismutase II showed the featureless absorption band in the range of 300-500 nm. The g values of electron paramagnetic resonance spectrum of superoxide dismutase II were gz = 4.762, gx = 4.072, and gy = 3.742, indicating iron-containing superoxide dismutase. Superoxide dismutase II uniquely contains 0.40 g-atom of iron per mole of monomer as well as 0.43 g-atom of zinc per mole of monomer. The immunological cross-reactivity between two isozymes was not found. Nickel-containing superoxide dismutase was widely distributed within the genus Streptomyces; however, iron- and zinc-containing superoxide dismutase was not found in S. albus and S. longisporoflavus, on the basis of the immunological cross-reactivity.

Spectral characterization and chemical modification of catalase-peroxidase from Streptomyces sp.

Catalase-peroxidase was purified to near homogeneity from Streptomyces sp. The enzyme was composed of two subunits with a molecular mass of 78 kDa and contained 1.05 mol of protoporphyrin IX/mol of dimeric protein. The absorption and resonance Raman spectra of the native and its cyano-enzyme were closely similar to those of other heme proteins with a histidine as the fifth ligand. However, the peak from tyrosine ring at approximately 1612 cm-1, which is unique in catalases, was not found in resonance Raman spectra of catalase-peroxidase. The electron paramagnetic resonance spectrum of the native enzyme revealed uniquely two sets of rhombic signals, which were converted to a single high spin, hexacoordinate species after the addition of sodium formate. Cyanide bound to the sixth coordination position of the heme iron, thereby converting the enzyme to a low spin, hexacoordinate species. The time-dependent inactivation of the enzyme with diethyl pyrocarbonate and its kinetic analysis strongly suggested the occurrence of histidine residue. From the above-mentioned spectroscopic results and chemical modification, it was deduced that the native enzyme is predominantly in the high spin, ferric form and has a histidine as the fifth ligand.