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Characterization of iron superoxide dismutase cDNAs from plants obtained by genetic complementation in Escherichia coli.

ABSTRACT: The inability of superoxide dismutase (SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1)-deficient mutants of Escherichia coli to grow aerobically on minimal medium can be restored by functional complementation with a heterologous SOD-encoding sequence. Based upon this property, a phenotypic selection system has been developed for the isolation of clones containing eukaryotic SOD cDNAs. cDNA expression libraries from both Nicotiana plumbaginifolia and Arabidopsis thaliana were transformed into a SOD-deficient E. coli strain by electroporation, and clones containing functional SODs were selected by growth on minimal medium. Analysis of these clones revealed the identity of cDNAs encoding the iron form of superoxide dismutase (FeSOD)--the first SODs of this type to be cloned from eukaryotes. The presence of this enzyme in these two divergent plant species challenges previous ideas that FeSOD is found in only a few plant families. In addition, these results show the potential for shotgun cloning of eukaryotic genes by complementation of bacterial mutants, particularly when it is combined with a highly efficient transformation method, such as electroporation.

SUBMITTER: Van Camp W

PROVIDER: S-EPMC55282 | biostudies-other | 1990 Dec

REPOSITORIES: biostudies-other

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Characterization of iron superoxide dismutase cDNAs from plants obtained by genetic complementation in Escherichia coli.

Van Camp W W Bowler C C Villarroel R R Tsang E W EW Van Montagu M M Inzé D D

Proceedings of the National Academy of Sciences of the United States of America 19901201 24

The inability of superoxide dismutase (SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1)-deficient mutants of Escherichia coli to grow aerobically on minimal medium can be restored by functional complementation with a heterologous SOD-encoding sequence. Based upon this property, a phenotypic selection system has been developed for the isolation of clones containing eukaryotic SOD cDNAs. cDNA expression libraries from both Nicotiana plumbaginifolia and Arabidopsis thaliana were transformed ...[more]

PMID: 2263641

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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.

Project description:The active-site structures of the oxidized and reduced forms of manganese-substituted iron superoxide dismutase (Mn(Fe)SOD) are examined, for the first time, using a combination of spectroscopic and computational methods. On the basis of electronic absorption, circular dichroism (CD), magnetic CD (MCD), and variable-temperature variable-field MCD data obtained for oxidized Mn(Fe)SOD, we propose that the active site of this species is virtually identical to that of wild-type manganese SOD (MnSOD), with both containing a metal ion that resides in a trigonal bipyramidal ligand environment. This proposal is corroborated by quantum mechanical/molecular mechanical (QM/MM) computations performed on complete protein models of Mn(Fe)SOD in both its oxidized and reduced states and, for comparison, wild-type (WT) MnSOD. The major differences between the QM/MM optimized active sites of WT MnSOD and Mn(Fe)SOD are a smaller (His)N-Mn-N(His) equatorial angle and a longer (Gln146(69))NH···O(sol) H-bond distance in the metal-substituted protein. Importantly, these modest geometric differences are consistent with our spectroscopic data obtained for the oxidized proteins and high-field electron paramagnetic resonance spectra reported previously for reduced Mn(Fe)SOD and MnSOD. As Mn(Fe)SOD exhibits a reduction midpoint potential (E(m)) almost 700 mV higher than that of MnSOD, which has been shown to be sufficient for explaining the lack of SOD activity displayed by the metal-subtituted species (Vance, C. K.; Miller, A. F. Biochemistry 2001, 40, 13079-13087), E(m)'s were computed for our experimentally validated QM/MM optimized models of Mn(Fe)SOD and MnSOD. These computations properly reproduce the experimental trend and reveal that the drastically elevated E(m) of the metal substituted protein stems from a larger separation between the second-sphere Gln residue and the coordinated solvent in Mn(Fe)SOD relative to MnSOD, which causes a weakening of the corresponding H-bond interaction in the oxidized state and alleviates steric crowding in the reduced state.

Dataset Information

Characterization of iron superoxide dismutase cDNAs from plants obtained by genetic complementation in Escherichia coli.

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Characterization of iron superoxide dismutase cDNAs from plants obtained by genetic complementation in Escherichia coli.

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