Substitution of histidine 30 by asparagine in manganese superoxide dismutase alters biophysical properties and supports proliferation in a K562 leukemia cell line.

We have generated a mutant of C. elegans manganese superoxide dismutase at histidine 30 by site-directed mutagenesis. The structure was solved at a resolution of 1.52 Å by X-ray crystallography (pdb: 6S0D). His30 was targeted, as it forms as a gateway residue at the top of the solvent access funnel to the active site, together with Tyr34. In the wild-type protein, these gateway residues are involved in the hydrogen-bonding network providing the protons necessary for the catalytic reaction at the metal center. However, biophysical characterization and cell viability experiments reveal that a mutation from histidine to asparagine in the H30N mutant modifies metal selectivity in the protein, favoring the uptake of iron over manganese in minimal media conditions, alters active-site coordination from the characteristic trigonal bipyramidal to octahedral geometry, and encourages cellular proliferation in K562 cells, when added exogenously to the cells.

Machine learning techniques for protein function prediction.

Proteins play important roles in living organisms, and their function is directly linked with their structure. Due to the growing gap between the number of proteins being discovered and their functional characterization (in particular as a result of experimental limitations), reliable prediction of protein function through computational means has become crucial. This paper reviews the machine learning techniques used in the literature, following their evolution from simple algorithms such as logistic regression to more advanced methods like support vector machines and modern deep neural networks. Hyperparameter optimization methods adopted to boost prediction performance are presented. In parallel, the metamorphosis in the features used by these algorithms from classical physicochemical properties and amino acid composition, up to text-derived features from biomedical literature and learned feature representations using autoencoders, together with feature selection and dimensionality reduction techniques, are also reviewed. The success stories in the application of these techniques to both general and specific protein function prediction are discussed.

Potential Therapeutic Applications of MnSODs and SOD-Mimetics.

Natural as well as synthetic antioxidants are constantly being investigated for their efficiency in combatting the effects of oxidative stress, which appears to be the responsible cause of several diseases, including cancer, central nervous system disorders, ischaemia-reperfusion disorders, cardiovascular conditions, and diabetes. Superoxide dismutases (SODs) constitute the ubiquitous antioxidant defences against oxidative stress that underlies numerous pathological conditions. Therefore, the development of therapeutics aimed at either delivering MnSOD more effectively to target tissues in the body in the form of MnSOD gene therapy, or the synthesis of molecules that mimic the activity of superoxide dismutase is constantly being explored. Classes that have been developed as SOD mimetics include the Mn-metalloporphyrins, Mn-cyclic polyamines, Mn-salen complexes, MnPLED derivatives as well as the nitroxides. Thus far, SOD mimetics have shown remarkable efficacy in several animal models suffering from oxidative stress injuries. A promising approach for the future of SOD and SOD mimic therapeutics appears to involve combination treatment of the antioxidants with radiotherapy or chemotherapy.

A Single Mutation is Sufficient to Modify the Metal Selectivity and Specificity of a Eukaryotic Manganese Superoxide Dismutase to Encompass Iron.

We have generated a site-directed mutant of the manganese superoxide dismutase SOD-3 of C.elegans (MnSOD-3) which modifies the metal specificity of the enzyme. While wild-type MnSOD-3 functions with manganese in the active site (3600 U mg-1 of protein) it has little or no activity when iron is incorporated. However, when histidine replaces glutamine 142 in the active site, the enzyme retains 50 % of its activity and becomes cambialistic for its metal cofactor exhibiting very similar specific activity with either manganese or iron.

Determination of metal content in superoxide dismutase enzymes by capillary electrophoresis†.

Superoxide dismutases are antioxidant scavenger enzymes that contain a metal cofactor (copper, zinc, iron, and manganese) in their active site. Metal content measurement is one of the essential steps to characterize enzyme biological activity. We have developed a capillary electrophoretic protocol for the determination of the metal content in superoxide dismutase enzymes. The background electrolyte containing 10 mM pyridine-2,6-dicarboxylic acid and 1 mM 1-methyl-3-tetradecylimidazolium chloride at pH 3.8 was optimized for on-column complexation of the above-mentioned metals. The minimum detectable levels of metals ranged from 0.3 to 1.2 µg/mL. The reliability of the method was checked by parallel quantitative determination of the metal content in superoxide dismutase enzymes by graphite furnace or flame atomic absorption spectrophotometry methods.

The structure of the Caenorhabditis elegans manganese superoxide dismutase MnSOD-3-azide complex.

C. elegans MnSOD-3 has been implicated in the longevity pathway and its mechanism of catalysis is relevant to the aging process and carcinogenesis. The structures of MnSOD-3 provide unique crystallographic evidence of a dynamic region of the tetrameric interface (residues 41-54). We have determined the structure of the MnSOD-3-azide complex to 1.77-Å resolution. Analysis of this complex shows that the substrate analog, azide, binds end-on to the manganese center as a sixth ligand and that it ligates directly to a third and new solvent molecule also positioned within interacting distance to the His30 and Tyr34 residues of the substrate access funnel. This is the first structure of a eukaryotic MnSOD-azide complex that demonstrates the extended, uninterrupted hydrogen-bonded network that forms a proton relay incorporating three outer sphere solvent molecules, the substrate analog, the gateway residues, Gln142, and the solvent ligand. This configuration supports the formation and release of the hydrogen peroxide product in agreement with the 5-6-5 catalytic mechanism for MnSOD. The high product dissociation constant k4 of MnSOD-3 reflects low product inhibition making this enzyme efficient even at high levels of superoxide.