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Molecular cloning, characterization, and expression in Escherichia coli of iron superoxide dismutase cDNA from Leishmania donovani chagasi.

ABSTRACT: A cDNA corresponding to superoxide dismutase (SOD; EC 1.15.1.1.) was isolated from a Leishmania donovani chagasi (L. d. chagasi) promastigote cDNA library, using PCR with a set of primers derived from conserved amino acids of manganese SODs (MnSODs) and iron SODs (FeSODs). Comparison of the deduced amino acid sequences with previously reported SOD amino acid sequences revealed that the L. d. chagasi 585-bp open reading frame had considerable homology with FeSODs and MnSODs. The highest homology was shared with prokaryotic FeSODs. The coding region of L. d. chagasi SOD cDNA has been expressed in fusion with glutathione-S-transferase, using an Escherichia coli mutant, QC779, lacking both MnSOD and FeSOD genes (sodA and sodB). Staining of native polyacrylamide gels for SOD activity of Leishmania crude lysate and the recombinant SOD revealed that both had SOD activity that was inactivated by 5 mM hydrogen peroxide but not by 2 mM potassium cyanide, which is indicative of FeSOD. The recombinant enzyme also protected E. coli mutant QC779 from paraquat toxicity. This indicated that the glutathione-S-transferase peptide does not interfere with the in vivo and in vitro activities of the recombinant SOD. Cross-species hybridization showed that FeSOD is highly conserved in the Leishmania genus. Interestingly, the hybridization pattern of the FeSOD gene(s) coincided with other classification schemes that divide Leishmania species into complexes. The cloning of FeSOD cDNA may contribute to the understanding of the role of SODs in Leishmania pathogenesis.

SUBMITTER: Ismail SO

PROVIDER: S-EPMC186154 | biostudies-other | 1994 Feb

REPOSITORIES: biostudies-other

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Molecular cloning, characterization, and expression in Escherichia coli of iron superoxide dismutase cDNA from Leishmania donovani chagasi.

Ismail S O SO Skeiky Y A YA Bhatia A A Omara-Opyene L A LA Gedamu L L

Infection and immunity 19940201 2

A cDNA corresponding to superoxide dismutase (SOD; EC 1.15.1.1.) was isolated from a Leishmania donovani chagasi (L. d. chagasi) promastigote cDNA library, using PCR with a set of primers derived from conserved amino acids of manganese SODs (MnSODs) and iron SODs (FeSODs). Comparison of the deduced amino acid sequences with previously reported SOD amino acid sequences revealed that the L. d. chagasi 585-bp open reading frame had considerable homology with FeSODs and MnSODs. The highest homology ...[more]

PMID: 8300222

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Project description:In Fe- and Mn-dependent superoxide dismutases (SODs), second-sphere residues have been implicated in precisely tuning the metal ion reduction potential to maximize catalytic activity (Vance, C. K.; Miller, A.-F. J. Am. Chem. Soc. 1998, 120, 461-467). In the present study, spectroscopic and computational methods were used to characterize three distinct Fe-bound SOD species that possess different second-coordination spheres and, consequently, Fe(3+/2+)reduction potentials that vary by approximately 1 V, namely, FeSOD, Fe-substituted MnSOD (Fe(Mn)SOD), and the Q69E FeSOD mutant. Despite having markedly different metal ion reduction potentials, FeSOD, Fe(Mn)SOD, and Q69E FeSOD exhibit virtually identical electronic absorption, circular dichroism, and magnetic circular dichroism (MCD) spectra in both their oxidized and reduced states. Likewise, variable-temperature, variable-field MCD data obtained for the oxidized and reduced species do not reveal any significant electronic, and thus geometric, variations within the Fe ligand environment. To gain insight into the mechanism of metal ion redox tuning, complete enzyme models for the oxidized and reduced states of all three Fe-bound SOD species were generated using combined quantum mechanics/molecular mechanics (QM/MM) geometry optimizations. Consistent with our spectroscopic data, density functional theory computations performed on the corresponding active-site models predict that the three SOD species share similar active-site electronic structures in both their oxidized and reduced states. By using the QM/MM-optimized active-site models in conjunction with the conductor-like screening model to calculate the proton-coupled Fe(3+/2+) reduction potentials, we found that different hydrogen-bonding interactions with the conserved second-sphere Gln (changed to Glu in Q69E FeSOD) greatly perturb the p K of the Fe-bound solvent ligand and, thus, drastically affect the proton-coupled metal ion reduction potential.

Dataset Information

Molecular cloning, characterization, and expression in Escherichia coli of iron superoxide dismutase cDNA from Leishmania donovani chagasi.

Publications

Molecular cloning, characterization, and expression in Escherichia coli of iron superoxide dismutase cDNA from Leishmania donovani chagasi.

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