Voyage of selenium from environment to life: Beneficial or toxic?

Selenium (Se), a naturally occurring metalloid, is an essential micronutrient for life as it is incorporated as selenocysteine in proteins. Although beneficial at low doses, Se is hazardous at high concentrations and poses a serious threat to various ecosystems. Due to this contrasting 'dual' nature, Se has garnered the attention of researchers wishing to unravel its puzzling properties. In this review, we describe the impact of selenium's journey from environment to diverse biological systems, with an emphasis on its chemical advantage. We describe the uneven distribution of Se and how this affects the bioavailability of this element, which, in turn, profoundly affects the habitat of a region. Once taken up, the subsequent incorporation of Se into proteins as selenocysteine and its antioxidant functions are detailed here. The causes of improved protein function due to the incorporation of redox-active Se atom (instead of S) are examined. Subsequently, the reasons for the deleterious effects of Se, which depend on its chemical form (organo-selenium or the inorganic forms) in different organisms are elaborated. Although Se is vital for the function of many antioxidant enzymes, how the pro-oxidant nature of Se can be potentially exploited in different therapies is highlighted. Furthermore, we succinctly explain how the presence of Se in biological systems offsets the toxic effects of heavy metal mercury. Finally, the different avenues of research that are fundamental to expand our understanding of selenium biology are suggested.

Unique functional insights into the antioxidant response of the cyanobacterial Mn-catalase (KatB).

KatB, a hexameric Mn-catalase, plays a vital role in overcoming oxidative and salinity stress in the ecologically important, N2-fixing cyanobacterium, Anabaena. The 5 N-terminal residues of KatB, which show a high degree of conservation in cyanobacteria, form an antiparallel ß-strand at the subunit interface of the KatB hexamer. In this study, the contribution of these N-terminal non-active site residues, towards the maintenance of the structure, biochemical properties, and redox balance was evaluated. Each N-terminal amino acid residue from the 2nd to the 7th position of KatB was individually mutated to Ala (to express KatBF2A/KatBF3A/KatBH4A/KatBK5E/KatBK6A/KatBE7A) or this entire 6 amino acid stretch was deleted (to yield KatBTrunc). All the above-mentioned KatB variants, along with the wild-type KatB protein (KatBWT), were overproduced in E. coli and purified. In comparison to KatBWT, the KatBF2A/KatBH4A/KatBTrunc proteins were less compact, more prone to chemical/thermal denaturation, and were unexpectedly inactive. KatBF3A/KatBK5E/KatBK6A showed biophysical/biochemical properties that were in between that of KatBWT and KatBF2A/KatBH4A/KatBTrunc. Surprisingly, KatBE7A was more thermostable with higher activity than KatBWT. On exposure to H2O2, E. coli expressing KatBWT/KatBE7A showed considerably reduced formation of ROS and increased survival than the other KatB variants. Utilizing the KatB structure, the molecular basis responsible for the altered stability/activity of the KatB mutants was delineated. This study demonstrates the physiological importance of the N-terminal ß-strand of Mn-catalases in combating H2O2 stress and shows that the non-active site residues can be used for rational protein engineering to develop Mn-catalases with improved characteristics.

Functional and mechanistic insights into the differential effect of the toxicant 'Se(IV)' in the cyanobacterium Anabaena PCC 7120.

Selenium, an essential trace element for animals, poses a threat to all forms of life above a threshold concentration. The ubiquitously present cyanobacteria, a major photosynthetic biotic component of aquatic and other ecosystems, are excellent systems to study the effects of environmental toxicants. The molecular changes that led to beneficial or detrimental effects in response to different doses of selenium oxyanion Se(IV) were analyzed in the filamentous cyanobacterium Anabaena PCC 7120. This organism showed no inhibition in growth up to 15 mg/L sodium selenite, but above this dose i.e. 20-100 mg/L of Se(IV), both growth and photosynthesis were substantially inhibited. Along with the increased accumulation of non-protein thiols, a consistent reduction in levels of ROS was observed at 10 mg/mL dose of Se(IV). High dose of Se(IV) (above 20 mg/L) enhanced endogenous reactive oxygen species (ROS)/lipid peroxidation, and decreased photosynthetic capability. Treatment with 100 mg/L Se(IV) downregulated transcription of several photosynthesis pathways-related genes such as those encoding photosystem I and II proteins, phycobilisome rod-core linker protein, phycocyanobilin, phycoerythrocyanin-associated proteins etc. Interestingly, at a dose range of 10-15 mg/L Se(IV), Anabaena showed an increase in PSII photosynthetic yield and electron transport rate (at PSII), suggesting improved photosynthesis. Se was incorporated into the Anabaena cells, and Se-enriched thylakoid membranes showed higher redox conductivity than the thylakoid membranes from untreated cells. Overall, the data supports that modulation of photosynthetic machinery is one of the crucial mechanisms responsible for the dose-dependent contrasting effect of Se(IV) observed in Anabaena.

Gazing into the remarkable world of non-heme catalases through the window of the cyanobacterial Mn-catalase 'KatB'.

Catalases, enzymes that decompose H2O2, are broadly categorized as heme catalases or non-heme catalases. The non-heme catalases are also known as Mn-catalases as they have Mn atoms in their active sites. However, unlike the well characterized heme-catalases, the study of Mn-catalases has gained importance only in the last few years. The filamentous, heterocystous, N2-fixing cyanobacterium Anabaena PCC 7120, shows the presence of two Mn-catalases, KatA and KatB, but lacks heme catalases. Of the two Mn-catalases, KatB, which is induced by salt/desiccation, plays a major role in overcoming salinity/oxidative stress. In this mini review, we have summarized the recent advances made in the field of Mn-catalases, particularly KatB, and have interpreted these results in the larger context of stress physiology. These aspects bring to the fore the distinctive biochemical/structural properties of Mn-catalases and furthermore highlight the in vivo importance of these enzymes in adapting to oxidative stresses.

Facile generation of a biotechnologically-relevant catalase showcases the efficacy of a blue-green algal biomass as a suitable bioresource for protein overproduction.

Here, we show the utility of a cyanobacterial biomass for overproduction and easy downstream processing of the thermostable protein KatB (a Mn-catalase). The nitrogen-fixing blue-green alga, Anabaena, was bioengineered to overexpress the KatB protein (An-KatB). Interestingly, pure An-KatB could be isolated from Anabaena by a simple physical process, obviating the need of expensive resins or chromatographic steps. An-KatB was an efficient H2O2-detoxifying protein that retained all the properties of Mn-catalases. Surprisingly, the purified An-KatB showed improved characteristics than the corresponding KatB (Ec-KatB) protein purified after over-expression in E. coli. An-KatB was unaffected by exposure to high temperature (85 °C), whereas a commercially procured heme-catalase showed an appreciable drop in activity beyond 50 °C. These data convincingly demonstrate the utility of Anabaena as a competent microbial bioresource for overproduction of proteins and further highlight the advantage of An-KatB over heme-catalases in bioprocesses where H2O2 is to be decomposed at elevated temperatures.

Novel molecular insights into the anti-oxidative stress response and structure-function of a salt-inducible cyanobacterial Mn-catalase.

KatB, a salt-inducible Mn-catalase, protects the cyanobacterium Anabaena from salinity/oxidative stress. In this report, we provide distinctive insights into the biological-biochemical function of KatB at the molecular level. Anabaena overexpressing the wild-type KatB protein (KatBWT) detoxified H2 O2 efficiently, showing reduced burden of reactive oxygen species compared with the strain overproducing KatBF2V (wherein F-2 is replaced by V). Correspondingly, the KatBWT protein also displayed several folds more activity than KatBF2V. Interestingly, the KatB variants with large hydrophobic amino acids (F/W/Y) were more compact, showed enhanced activity, and were resistant to thermal/chemical denaturation than variants with smaller residues (G/A/V) at the second position. X-ray crystallography-based analysis showed that F-2 was required for appropriate interactions between two subunits. These contacts provided stability to the hexamer, making it more compact. F-2, through its interaction with F-66 and W-43, formed the proper hydrophobic pocket that held the active site together. Consequently, only residues that supported activity (i.e., F/Y/W) were selected at the second position in Mn-catalases during evolution. This study (a) demonstrates that modification of nonactive site residues can alter the response of catalases to environmental stress and (b) has expanded the scope of amino acids that can be targeted for rational protein engineering in plants.

Molecular basis of function and the unusual antioxidant activity of a cyanobacterial cysteine desulfurase.

Cysteine desulfurases, which supply sulfur for iron-sulfur cluster biogenesis, are broadly distributed in all phyla including cyanobacteria, the progenitors of plant chloroplasts. The SUF (sulfur utilization factor) system is responsible for Fe-S cluster biosynthesis under stress. The suf operon from cyanobacterium Anabaena PCC 7120 showed the presence of a cysteine desulfurase, sufS (alr2495), but not the accessory sulfur-accepting protein (SufE). However, an open reading frame (alr3513) encoding a SufE-like protein (termed AsaE, Anabaena sulfur acceptor E) was found at a location distinct from the suf operon. The purified SufS protein existed as a pyridoxal 5' phosphate (PLP)-containing dimer with a relatively low desulfurase activity. Interestingly, in the presence of the AsaE protein, the catalytic efficiency of this reaction increased 10-fold. In particular, for sulfur mobilization, the AsaE protein partnered only SufS and not other cysteine desulfurases from Anabaena. The SufS protein was found to physically interact with the AsaE protein, demonstrating that AsaE was indeed the missing partner of Anabaena SufS. The conserved cysteine of the SufS or the AsaE protein was essential for activity but not for their physical association. Curiously, overexpression of the SufS protein in Anabaena caused reduced formation of reactive oxygen species on exposure to hydrogen peroxide (H2O2), resulting in superior oxidative stress tolerance to the oxidizing agent when compared with the wild-type strain. Overall, the results highlight the functional interaction between the two proteins that mediate sulfur mobilization, in the cyanobacterial SUF pathway, and further reveal that overexpression of SufS can protect cyanobacteria from oxidative stress.

Cyanobacterial Mn-catalase 'KatB': Molecular link between salinity and oxidative stress resistance.

Catalases are ubiquitous enzymes that detoxify H2O2 in virtually all organisms exposed to oxygen. The filamentous, nitrogen-fixing cyanobacterium, Anabaena PCC 7120, shows the presence of 2 genes (katA and katB) that encode Mn-catalases. We have recently shown that pre-treatment of Anabaena with NaCl causes substantial induction of the KatB protein, which consequently leads to increased oxidative stress resistance in that cyanobacterium. Interestingly, when compared to the wild-type, the katB mutant shows decreased growth and impaired photosynthetic activity in the presence of NaCl. Furthermore, the NaCl-treated katB mutant is extremely sensitive to H2O2. In this study, the ultrastructural changes occurring in the katB mutant and the wild-type Anabaena cells are analyzed to understand the cellular basis of the above-mentioned protective phenomena. Other data show that a wide variety of osmolytes induce katB expression in Anabaena, indicating that katB is a genuine osmo-inducible gene. These results have important biotechnological implications for the development of novel cyanobacterial biofertilzers and transgenic plants with improved resistance to salinity.

KatB, a cyanobacterial Mn-catalase with unique active site configuration: Implications for enzyme function.

Manganese catalases (Mn-catalases), a class of H2O2 detoxifying proteins, are structurally and mechanistically distinct from the commonly occurring catalases, which contain heme. Active site of Mn-catalases can serve as template for the synthesis of catalase mimetics for therapeutic intervention in oxidative stress related disorders. However, unlike the heme catalases, structural aspects of Mn-catalases remain inadequately explored. The genome of the ancient cyanobacterium Anabaena PCC7120, shows the presence of two Mn-catalases, KatA and KatB. Here, we report the biochemical and structural characterization of KatB. The KatB protein (with a C-terminal his-tag) was over-expressed in Escherichia coli and purified by affinity chromatography. On the addition of Mn(2+) to the E. coli growth medium, a substantial increase in production of the soluble KatB protein was observed. The purified KatB protein was an efficient catalase, which was relatively insensitive to inhibition by azide. Crystal structure of KatB showed a hexameric assembly with four-helix bundle fold, characteristic of the Ferritin-like superfamily. With canonical Glu4His2 coordination geometry and two terminal water ligands, the KatB active site was distinctly different from that of other Mn-catalases. Interestingly, the KatB active site closely resembled the active sites of ruberythrin/bacterioferritin, bi-iron members of the Ferritin-like superfamily. The KatB crystal structure provided fundamental insights into the evolutionary relationship within the Ferritin-like superfamily and further showed that Mn-catalases can be sub-divided into two groups, each with a distinct active site configuration.

A Salt-Inducible Mn-Catalase (KatB) Protects Cyanobacterium from Oxidative Stress.

Catalases, enzymes that detoxify H2O2, are widely distributed in all phyla, including cyanobacteria. Unlike the heme-containing catalases, the physiological roles of Mn-catalases remain inadequately characterized. In the cyanobacterium Anabaena, pretreatment of cells with NaCl resulted in unusually enhanced tolerance to oxidative stress. On exposure to H2O2, the NaCl-treated Anabaena showed reduced formation of reactive oxygen species, peroxides, and oxidized proteins than the control cells (i.e. not treated with NaCl) exposed to H2O2. This protective effect correlated well with the substantial increase in production of KatB, a Mn-catalase. Addition of NaCl did not safeguard the katB mutant from H2O2, suggesting that KatB was indeed responsible for detoxifying the externally added H2O2. Moreover, Anabaena deficient in KatB was susceptible to oxidative effects of salinity stress. The katB gene was strongly induced in response to osmotic stress or desiccation. Promoter-gfp analysis showed katB to be expressed only in the vegetative cells but not in heterocysts. Biochemically, KatB was an efficient, robust catalase that remained active in the presence of high concentrations of NaCl. Our findings unravel the role of Mn-catalase in acclimatization to salt/oxidative stress and demonstrate that the oxidative stress resistance of an organism can be enhanced by a simple compound such as NaCl.

Redox-dependent chaperone/peroxidase function of 2-Cys-Prx from the cyanobacterium Anabaena PCC7120: role in oxidative stress tolerance.

BACKGROUND: Cyanobacteria, progenitors of plant chloroplasts, provide a suitable model system for plants to study adaptation towards different abiotic stresses. Genome of the filamentous, heterocystous, nitrogen-fixing cyanobacterium Anabaena PCC7120 harbours a single gene (alr4641) encoding a typical 2-Cys-Peroxiredoxins (2-Cys-Prxs). 2-Cys-Prxs are thiol-based peroxidases that also function as molecular chaperones in plants and other systems. The Alr4641 protein from Anabaena PCC7120 shows high level biochemical similarities with the plant 2-Cys-Prx. The physiological role played by the Alr4641 protein in Anabaena was addressed in this study. RESULTS: In Anabaena PCC7120, alr4641 transcript /Alr4641 protein was induced in response to abiotic stresses and its promoter was active in the vegetative cells as well as heterocysts. The wild-type Alr4641 protein or Alr4641 lacking the peroxidatic cysteine (Alr4641C56S) or the resolving cysteine (Alr4641C178S) existed as higher oligomers in their native form. The wild-type or the mutant Alr4641 proteins showed similar chaperone activity, but only the wild-type protein exhibited peroxidase activity indicating that unlike peroxidase activity, chaperone activity was not dependent on cysteines. In contrast to other 2-Cys-Prxs, chaperone/peroxidase activity of Alr4641 was dependent on its redox state and not oligomerization status. Alr4641 could protect plasmid DNA from oxidative damage and physically associate with NADPH-dependent thioredoxin reductase (NTRC). Like 2-Cys-Prxs from plants (e.g. rice), Alr4641 could detoxify various peroxides using NTRC as reductant. On exposure to H2O2, recombinant Anabaena PCC7120 strain over-expressing Alr4641 (An4641+) showed reduced content of reactive oxygen species (ROS), intact photosynthetic functions and consequently better survival than the wild-type Anabaena PCC7120, indicating that Alr4641 can protect Anabaena from oxidative stress. CONCLUSIONS: The peroxidase/chaperone function of Alr4641, its inherent transcriptional/translational induction under different abiotic stresses and localization in both vegetative cells and heterocysts could be an adaptive strategy to battle various oxidative stresses that Anabaena encounters during its growth. Moreover, the recombinant Anabaena strain over expressing Alr4641 showed higher resistance to oxidative stress, suggesting its potential to serve as stress-tolerant biofertilizers in rice fields.

Purification, crystallization and preliminary crystallographic analysis of KatB, a manganese catalase from Anabaena PCC 7120.

Catalases are enzymes that play an important role in the detoxification of hydrogen peroxide (H2O2) in aerobic organisms. Among catalases, haem-containing catalases are ubiquitously distributed and their enzymatic mechanism is very well understood. On the other hand, manganese catalases that contain a bimanganese core in the active site have been less well characterized and their mode of action is not fully understood. The genome of Anabaena PCC 7120 does not show the presence of a haem catalase-like gene; instead, two ORFs encoding manganese catalases (Mn-catalases) are present. Here, the crystallization and preliminary X-ray crystallographic analysis of KatB, one of the two Mn-catalases from Anabaena, are reported. KatB was crystallized using the hanging-drop vapour-diffusion method with PEG 400 as a precipitant and calcium acetate as an additive. Diffraction data were collected in-house on an Agilent SuperNova system using a microfocus sealed-tube X-ray source. The crystal diffracted to 2.2 Šresolution at 100 K. The tetragonal crystal belonged to space group P4(1)2(1)2 (or enantiomer), with unit-cell parameters a = b = 101.87, c = 138.86 Å. Preliminary X-ray diffraction analysis using the Matthews coefficient and self-rotation function suggests the presence of a trimer in the asymmetric unit.