Conversion of the metal-specific activity of Escherichia coli Mn-SOD by site-directed mutagenesis of Gly165Thr

Glycine 165, which is located near the active site metal, is mostly conserved in aligned amino acid sequences of manganese-containing superoxide dismutase (Mn-SOD) proteins, but is substituted to threonine in most iron-containing SODs (Fe-SODs). Because threonine 165 is located between Trp128 and Trp130, and Trp128 is one of the metal-surrounding aromatic amino acids, the conversion of this amino acid may affect the metal-specific activity of Escherichia coli Mn-SOD. In order to clarify this possibility, we prepared a mutant of E. coli Mn-SOD with the replacement of Gly165 by Thr. The ratio of the specific activities of Mn- to Fe-reconstituted enzyme increased from 0.006 in the wild-type to 0.044 in the mutant SOD; therefore, the metal-specific SOD was converted to a metal-tolerant SOD. The visible absorption spectra of the Fe- and Mn-reconstituted mutant SODs indicated the loss of Mn-SOD character. It was concluded that Gly at position 165 plays a catalytic role in maintaining the integrity of the metal specificity of Mn-SOD.     

Cloning and characterization of a thermostable superoxide dismutase from the thermophilic bacterium Rhodothermus sp. XMH10

A superoxide dismutase (SOD) gene was cloned from the thermophilic bacterium Rhodothermus sp. XMH10 for the first time and highly expressed in Escherichia coli. The Rhodothermus sp. XMH10 SOD (RhSOD) gene encodes 209 amino acids with a putative molecular weight of 23.6 kDa and a pI value of 5.53. The recombinant RhSOD was detected to be an iron type SOD and existed as a dimer on its natural status. Experiments revealed that this RhSOD showed high activity at 50–70 °C and pH 5.0. Compared to SODs from other thermophiles, it was highly thermostable, maintaining more than 90% of its activity after incubation at 70 °C for 12 h, only totally inactivated after more than 4-h incubation at 80 °C. It also showed much higher resistance to KCN, NaN₃ and H₂O₂ as compared to other SODs. Xin Wang and Haijie Yang contribute to this work equally.     

Engineering a highly thermostable and stress tolerant superoxide dismutase by N-terminal modification and metal incorporation

Thermophilic or hyperthermophilic SODs (superoxide dismutase) usually offer substantial biotechnological advantages over mesophilic SODs. Previously a 244-amino acid N-terminal domain (NTD) from a heatresistant SOD of Geobacillus thermodenitrificans NG80-2 was discovered and demonstrated to be able to confer thermostability to homologous mesophilic SODs, which revealed a new type of heat resistance mechanism. To further improve the heat resistance and stress tolerance of thermophilic cambialistic superoxide dismutase (Fe/Mn- SOD _Ap ) from Aeropyrum pernix K1 through metal incorporation and fusion with the newly found peptide NTD for broadening its industrial application, the wildtype SOD _Ap and NTD-fused ntdSOD _Ap were expressed in E. coli BL21 and incorporated with metal cofactors by two ways. Recombinant fusion SOD obtained by in vitro reconstitution (Mn-rec ntdSOD _Ap ) exhibited improved optimum temperature at 70°C and dramatically enhanced thermostability especially at 110°C with enhanced pH stability from 4 to 10 and higher tolerance for denaturants and organic media than Mn-rec SOD _Ap . To the best of our knowledge, Mn-rec ntdSOD _Ap could be the most heat resistant SOD. In addition, metal incorporation of SOD _Ap and ntdSOD _Ap via in vivo modification have been developed and proved to be more practical for industrial use. These results indicate that fusion with NTD along with metal incorporation can generate superimposed effect and be applied to enhance the stability of cambialistic thermophilic SODs, thus providing a universal and convenient bioengineering method for generating extremely stable SODs.     

The Positive Charge at Position 189 IS Essential for the Catalytic Activity of Iron-and Manganese-Containing Superoxide Dismutases

We have previously shown (C.L. Borders, Jr. el al., (1989) Archives of Biochemistry and Eiaphysics. 268, 74–80) that the iron-containing (FeSOD) and manganese-containing (MnSOD) superoxide dismutases from Eschericliia coli are extensively (≥98%) inactivated by treatment with phenylglyoxal. an arginine-specific reagent. Examination of the published primary sequences of these two enzymes shows that Arg-189 is the only conserved arginine. This arginine is also conserved in the three additional FeSODs and seven of the eight additional MnSODs sequenced to date, with the only exception king the MnSOD from Saccharomyces cerevisiae, in which it is conservatively replaced by lysine. Treatment of S. cerevisiae MnSOD with phenylglyoxal under the same conditions used for the E. coli enzymes gives very little inactivation. However, treatment with low levels of 2.4.6-trinitrobenzenesulfonate (TNBS) and acetic anhydride, two lysine-selective reagents that cause a maximum of 65–80% inactivation of the E. coli SODs, gives complete inactivation of the yeast enzyme. Total inactivation of yeast MnSOD with TNBS correlates with the modification of approximately 5 lysines per subunit, whereas 6–7 lysines per subunit are acylated with acetic anhydride on complete inactivation. It appears that the positive charge contributed by residue 189. lysine in yeast MnSOD and arginine in all other SODs. may be critical for the catalytic activity or MnSODs and FeSODs.     

Indolepyruvate ferredoxin oxidoreductase from Pyrococcus sp. KOD1 possesses a mosaic structure showing features of various oxidoreductases

Indolepyruvate ferredoxin oxidoreductase (IOR) catalyzes the oxidative decarboxylation of arylpyruvates. Gene cloning and sequencing analysis of the IOR gene from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was performed. Two genes, iorA and iorB, encoding α and β subunits of IOR were found to be tandemly arranged, which suggests that gene expression is translationaly coupled. Sequence analysis showed the C-terminal region of the α subunit to have a typical ferredoxin-type [4Fe-4S] cluster motif (CXXCXXCXXCXXXCP), which is similar to that present in the δ subunits of other oxidoreductases such as pyruvate ferredoxin oxidoreductase (POR) and 2-ketoisovalerate ferredoxin oxidoreductase (VOR). We suggest that the α subunit of KOD1-IOR has a mosaic structure composed of features characteristic of the α, β and δ subunits from POR and VOR. KOD1-IOR was overproduced in anaerobically incubated Escherichia coli cells and the crude enzyme was extracted under anaerobic conditions. The optimal temperature for activity of recombinant IOR was 70° C and the half-life of this enzyme in the presence of air was 15 min at 25° C. Received: 25 September 1996 / Accepted: 20 December 1996     

Thermostability, oligomerization and DNA-binding properties of the regulatory protein ArgR from the hyperthermophilic bacterium Thermotoga neapolitana

The hexameric regulatory protein ArgR formed by arginine-mediated dimerization of identical trimers governs the expression of genes required for arginine metabolism and some other genes in mesophilic and moderately thermophilic bacteria. We have cloned the argR gene from two hyperthermophilic bacteria of the genus Thermotoga. The two-domain ArgR proteins encoded by T. neapolitana and T. maritima share a low degree of sequence similarity with other bacterial arginine repressors. The ArgR protein from T. neapolitana binds to an operator located just upstream of its coding sequence and, therefore, the argR gene may be autoregulated. The protein has extremely high intrinsic thermostability and tolerance to urea. Moreover, its binding to target DNA increases the melting temperature by approximately 15° C. The formation of oligomeric ArgR-DNA complexes is a function of protein concentration, with hexameric complexes being favoured at higher concentrations. In the presence of arginine the hyperthermophilic ArgR protein binds to its own operator, argRo, only by forming hexamer ArgR-DNA complexes, whereas both trimer-DNA and hexamer-DNA complexes are detected in the absence of arginine. However, the affinity of T. neapolitana ArgR for DNA has been found to be higher for a mixture of trimers and non-bound hexamers than for arginine-bound hexamers. Our data indicate that genes for arginine biosynthesis are clustered in a putative operon, which could also be regulated by the ArgR protein, in the hyperthermophilic host. Received: 19 July 1999 / Accepted: 4 November 1999     

Superoxide dismutases: ancient enzymes and new insights

Superoxide dismutases (SODs) catalyze the de toxification of superoxide. SODs therefore acquired great importance as O(2) became prevalent following the evolution of oxygenic photosynthesis. Thus the three forms of SOD provide intriguing insights into the evolution of the organisms and organelles that carry them today. Although ancient organisms employed Fe-dependent SODs, oxidation of the environment made Fe less bio-available, and more dangerous. Indeed, modern lineages make greater use of homologous Mn-dependent SODs. Our studies on the Fe-substituted MnSOD of Escherichia coli, as well as redox tuning in the FeSOD of E. coli shed light on how evolution accommodated differences between Fe and Mn that would affect SOD performance, in SOD proteins whose activity is specific to one or other metal ion.     

Cloning and expression of a gene encoding cyanidase from Pseudomonas stutzeri AK61

The gene coding for cyanidase, which catalyzes the hydrolysis of cyanide to formate and ammonia, was cloned from chromosomal DNA of Pseudomonas stutzeri AK61 into Escherichia coli. The cyanidase gene consisted of an open reading frame of 1004 bp, and it was predicted that cyanidase was composed of 334 amino acids with a calculated molecular mass of 37 518 Da. The amino acid sequence of cyanidase showed a 35.1% and 26.4% homology to aliphatic nitrilase from Rhodococcus rhodochrous K22 and cyanide hydratase from Fusarium lateritium, respectively. A unique cysteine residue of aliphatic nitrilase, which was suggested to play an essential role in the catalytic activity, was conserved in cyanidase. The active form of cyanidase was successfully expressed by a DNA clone containing the cyanidase gene in E.coli. Its productivity was approximately 230 times larger than that of P. stutzeri AK61. The characteristics of the expressed cyanidase, including optimum pH, optimum temperature, Michaelis constant (K _m) for cyanide and specific activity, were similar to those of the native enzyme from P. stutzeri AK61. Received: 24 October 1997 / Received last revision: 17 March 1998 / Accepted: 20 March 1998     

Involvement of manganese peroxidase in the transformation of macromolecules from low-rank coal by Phanerochaete chrysosporium

Manganese peroxidase (Mn peroxidase) catalyses the oxidation of Mn(II) to Mn(III), a diffusible non-specific oxidant likely to be involved in the transformation of polyphenolic macromolecules from brown coal by the white-rot fungus Phanerochaete chrysosporium. We report here that solubilised macromolecules from Morwell brown coal were depolymerised by Mn(III) ions when incubated under hyperbaric O₂. However, under N₂ or air they were polymerised, suggesting that net depolymerisation by Mn(III) requires molecular oxygen to inhibit coupling of coal radicals. Coal macromolecules were also polymerised when separated by a semipermeable membrane from a culture of P. chrysosporium or from a solution of Mn peroxidase, Mn(II) and H₂O₂, probably by Mn(III) crossing the membrane. In oxygenated cultures in which Mn peroxidase␣was up-regulated by Mn(II), the extent of depolymerisation correlated with cumulative Mn peroxidase activity suggesting that Mn-peroxidase-generated Mn(III) has a central role in initial depolymerisation of coal molecules in vivo. However, mutant ME446-B17-1, which produces Mn peroxidase but not lignin peroxidase, polymerised coal macromolecules in oxygenated cultures. In sum, it appears Mn peroxidase can both polymerise and depolymerise brown coal macromolecules and that, in vivo, both hyperbaric O₂ and lignin peroxidase are also required to force net depolymerisation to products assimilable by cells. Received: 4 September 1997 / Received revision: 29 January 1998 / Accepted: 30 January 1998     

Unique active site formation in a novel galactose 1-phosphate uridylyltransferase from the hyperthermophilic archaeon Pyrobaculum aerophilum

A gene encoding galactose 1-phosphate uridylyltransferase (GalT) was identified in the hyperthermophilic archaeon Pyrobaculum aerophilum. The gene was overexpressed in Escherichia coli, after which its product was purified and characterized. The expressed enzyme was highly thermostable and retained about 90% of its activity after incubation for 10 minutes at temperatures up to 90°C. Two different crystal structures of P. aerophilum GalT were determined: the substrate-free enzyme at 2.33 Å and the UDP-bound H140F mutant enzyme at 1.78 Å. The main-chain coordinates of the P. aerophilum GalT monomer were similar to those in the structures of the E. coli and human GalTs, as was the dimeric arrangement. However, there was a striking topological difference between P. aerophilum GalT and the other two enzymes. In the E. coli and human enzymes, the N-terminal chain extends from one subunit into the other and forms part of the substrate-binding pocket in the neighboring subunit. By contrast, the N-terminal chain in P. aerophilum GalT extends to the substrate-binding site in the same subunit. Amino acid sequence alignment showed that a shorter surface loop in the N-terminal region contributes to the unique topology of P. aerophilum GalT. Structural comparison of the substrate-free enzyme with UDP-bound H140F suggests that binding of the glucose moiety of the substrate, but not the UDP moiety, gives rise to a large structural change around the active site. This may in turn provide an appropriate environment for the enzyme reaction.     

Quantitative evaluation of metal ion binding to PvuII restriction endonuclease

19 F NMR spectroscopy have been applied to evaluate metal ion binding by the representative PvuII endonuclease in the absence of substrate. In separate experiments, ITC data demonstrate that PvuII endonuclease binds 2.16 Mn(II) ions and 2.05 Ca(II) metal ions in each monomer active site with K _d values of  ≈ 1 mM. While neither calorimetry nor protein NMR spectroscopy is directly sensitive to Mg(II) binding to the enzyme, Mn(II) competes with Mg(II) for common sites(s) on PvuII endonuclease. Substitution of the conserved active site carboxylate Glu68 with Ala resulted in a loss of affinity for both equivalents of both Ca(II) and Mn(II). Interestingly, the active site mutant D58A retained an affinity for Mn(II) with K _d  ≈ 2 mM. Mn(II) paramagnetic broadening in ¹⁹F spectra of wild-type and mutant 3-fluorotyrosine PvuII endonucleases are consistent with ITC results. Chemical shift analysis of 3-fluorotyrosine mutant enzymes is consistent with a perturbed conformation for D58A. Therefore, free PvuII endonuclease binds metal ions, and metal ion binding can precede DNA binding. Further, while Glu68 is critical to metal ion binding, Asp58 does not appear to be critical to the binding of at least one metal ion and appears to also have a role in structure. These findings provide impetus for exploring the roles of multiple metal ions in the structure and function of this representative endonuclease. Received: 30 March 1999 / Accepted: 28 September 1999     

Mechanism of dithionite reduction of the R2 protein of E. coli and mouse ribonucleotide reductase: evidence for reduction of FeIII 2 prior to the tyrosyl radical

 Dithionite has been found to reduce directly (without mediators) the Escherichia coli R2 subunit of ribonucleotide reductase. With dithionite (∼10 mM) in large excess, the reaction at 25  °C is complete in ∼10 h. Preparations of E. coli R2 have an Fe^III ₂ (met-R2) component in this work at ∼40% levels, alongside the fully active enzyme Fe^III ₂ . . . Tyr*, which has a tyrosyl radical at Tyr-122. In the pH range studied (7–8) the kinetics are biphasic. Rate laws for both phases give [S₂O₄ ^2–] and not [S₂O₄ ^2–]^1/2 dependencies, and saturation kinetics are observed for the first time in R2 studies. No dependence on pH was detected. The kinetics (25  °C) of the first phase are reproduced in separate experiments using only met-R2, with association of S₂O₄ ^2– to met-R2, K=330 M^–1, occurring prior to electron transfer, k _et=4.8×10^–4 s^–1, I=0.100 M (NaCl). The second phase assigned to the reaction of Fe^III ₂ . . . Tyr* with S₂O₄ ^2– gives K=800 M^–1 and k _et=5.6×10^–5 s^–1. Bearing in mind the substantially smaller reduction potential for Fe^III ₂ compared to Tyr*, this is a quite remarkable finding, with implications similar to those already reported for the reaction of R2 with hydrazine, but with additional information provided by the saturation kinetics. The similarity in rates for the two phases (∼fourfold difference) suggests that reduction of Fe^III ₂ is occurring in both cases, and since S₂O₄ ^2– is involved a two-equivalent change is proposed with the formation of Fe^II ₂ . . . Tyr* in the case of active R2. As a sequel to the second phase, intramolecular reduction of the strongly oxidising Tyr* by the Fe^II ₂ is rapid, and further decay of Fe^IIFe^III is also fast. There is no stable mouse met-R2 form, and the single-phase reaction with dithionite gives saturation kinetics with K=208 M^–1 and k _et=1.7±10^–3 s^–1. Mechanistic implications, including the applicability of a pathway for electron transfer via Fe_A, are considered. Received: 25 February 1998 / Received: 20 August 1998     

Regulation of replication of λ phage and λ plasmid DNAs at low temperature

It was previously demonstrated that while lysogenic development of bacteriophage λ in Escherichia coli proceeds normally at low temperature (20–25° C), lytic development is blocked under these conditions owing to the increased stability of the phage CII protein. This effect was proposed to be responsible for the increased stimulation of the p _E promoter, which interferes with expression of the replication genes, leading to inhibition of phage DNA synthesis. Here we demonstrate that the burst size of phage λcIb2, which is incapable of lysogenic development, increases gradually over the temperature range from 20 to 37° C, while no phage progeny are observed at 20° C. Contrary to previous reports, it is possible to demonstrate that p _E promoter activation by CII may be more efficient at lower temperature. Using density-shift experiments, we found that phage DNA replication is completely blocked at 20° C. Phage growth was also inhibited in cells overexpressing cII, which confirms that CII is responsible for inhibition of phage DNA replication. Unexpectedly, we found that replication of plasmids derived from bacteriophage λ is neither inhibited at 20° C nor in cells overexpressing cII. We propose a model to explanation the differences in replication observed between λ phage and λ plasmid DNA at low temperature. Received: 30 December 1997 / Accepted: 25 February 1998     

Cloning and characterization of a new manganese superoxide dismutase from deep-sea thermophile Geobacillus sp. EPT3

A new gene encoding a superoxide dismutase (SOD) was identified from a thermophile Geobacillus sp. EPT3 isolated from a deep-sea hydrothermal field in east Pacific. The open reading frame of this gene encoded 437 amino acid residues. It was cloned, overexpressed in Escherichia coli (DE3), and the recombinant protein was purified to homogeneity. Geobacillus sp. EPT3 SOD was of the manganese-containing SOD type, as judged by the insensitivity of the recombinant enzyme to both KCN and H₂O₂, and the activity analysis of Fe or Mn reconstituted SODs by polyacrylamide gel electrophoresis. The recombinant SOD was determined to be a homodimer with monomeric molecular mass of 59.0 kDa. In comparison with other Mn–SODs, the manganese-binding sites are conserved in the sequence (His260, His308, Asp392, His396). The recombinant enzyme had high thermostability at 50 °C. It retained 57 % residual activity after incubation at 90 °C for 1 h, which indicated that this SOD was thermostable. The enzyme also showed striking stability over a wide range of pH 5.0–11.0. At tested conditions, the recombinant SOD from Geobacillus sp. EPT3 showed a relatively good tolerance to some inhibitors, detergents, and denaturants, such as β-mercaptoethanol, dithiothreitol, phenylmethylsulfonyl fluoride, Chaps, Triton X-100, urea, and guanidine hydrochloride.     

Depolymerization of low-rank coal by extracellular fungal enzyme systems. III.In vitro depolymerization of coal humic acids by a crude preparation of manganese peroxidase from the white-rot fungus Nematoloma frowardii b19

The in vitro depolymerization of humic acids derived from German lignite (low-rank coal, brown coal) was studied using a manganese peroxidase preparation from the white-rot fungus Nematoloma frowardii b19. The H₂O₂ required was continuously generated by glucose oxidase. Mn peroxidase depolymerized high-molecular-mass humic acids by forming fulvic-acid-like compounds. The depolymerization process was accompanied by the decolorization of the dark-brown humic acid fraction soluble in alkaline solutions (decrease in absorbance at 450 nm) and by the yellowish coloring of the fraction of acid-soluble fulvic-acid-like compounds (increase in absorbance at 360 nm). The Mn peroxidase of N. frowardii b19 has been proved to be highly stable; even after an in vitro reaction time of 7 days in the presence of humic acids, less than 10% loss in total oxidizing activity was detectable. Received: 16 September 1996 / Received revision: 16 December 1996 / Accepted: 20 December 1996     

Enhancement of superoxide dismutase activity in the leaves of white clover (Trifolium repens L.) in response to polyethylene glycol-induced water stress

Effects of polyethylene glycol (PEG)-induced water stress on the activities of total leaf superoxide dismutase (SOD) and chloroplast SOD (including thylakoid-bound SOD and stroma SOD) are described in white clover (Trifolium repens L.) grown in solution culture from rooted cuttings. Both leaf SOD and chloroplast SOD activities were markedly enhanced with increasing concentration of PEG stress, generating osmotic potentials around the roots 0, −0.5, −1.0, −1.5 MPa. The effects increased with time up to 72 h. Chloroplast Fe-containing SOD represented about 30% of the total leaf SOD activity in the control plants and a significant increase in chloroplast SOD activity was found during the stress period. This accounted for about 35.5–71.1% of the total leaf SOD activity. The proportion of chloroplast SOD in total leaf SOD not only increased with the decreasing of osmotic potential, but also increased with incubation time. Furthermore, the increase in thylakoid-bound SOD activity was much higher than that of stroma SOD in chloroplast of plants under water stress. The enhanced chloroplastic SOD activity, especially thylakoid-bound SOD activity, demonstrated in Trifolium repens suggests that Fe-SOD located in chloroplasts play a more important role than cytosolic Cu/Zn-containing SODs in scavenging O₂ ⁻.     

Isolation and characterization of pyrimidine auxotrophs, and molecular cloning of the pyrE gene from the hyperthermophilic archaeon Pyrococcus abyssi

Uracil auxotrophic mutants of the hyperthermophilic archaeon Pyrococcus abyssi were isolated by screening for resistance to 5-fluoro-orotic acid (5-FOA). Wild-type strains were unable to grow on medium containing 5-FOA, whereas mutants grew normally. Enzymatic assays of extracts from wild-type P. abyssi and from pyrimidine auxotrophs demonstrated that the mutants are deficient in orotate phosphoribosyltransferase (PyrE) and/or orotidine-5′-monophosphate decarboxylase (PyrF) activity. The pyrE gene of wild-type P. abyssi and one of its mutant derivatives were cloned and sequenced. This pyrE gene could serve as selectable marker for the development of gene manipulation systems in archaeal hyperthermophiles. Received: 29 March 1999 / Accepted: 25 May 1999     

Effect of ammonia on the anaerobic degradation of protein by a mesophilic and thermophilic biowaste population

The influence of ammonia on the anaerobic degradation of peptone by mesophilic and thermophilic populations of biowaste was investigated. For peptone concentrations from 5 g l⁻¹ to 20 g l⁻¹ the mesophilic population revealed a higher rate of deamination than the thermophilic population, e.g. 552 mg l⁻¹ day⁻¹ compared to 320 mg l⁻¹ day⁻¹ at 10 g l⁻¹ peptone. The final degree of deamination of the thermophilic population was, however, higher: 102 compared to 87 mg NH₃/g peptone in the mesophilic cultures. If 0.5–6.5 g l⁻¹ ammonia was added to the mesophilic biowaste cultures, deamination of peptone, degradation of its chemical oxygen demand (COD) and formation of biogas were increasingly inhibited, but no hydrogen was formed. The thermophilic biowaste cultures were most active if around 1 g ammonia l⁻¹ was present. Deamination, COD degradation and biogas production decreased at lower and higher ammonia concentrations and hydrogen was formed in addition to methane. Studies of the inhibition by ammonia of peptone deamination, COD degradation and methane formation revealed a K _i (50%) for NH₃ of 92, 95 and 88 mg l⁻¹ at 37 °C and 251, 274 and 297 mg l⁻¹ at 55 °C respectively. This indicated that the thermophilic flora tolerated significantly more NH₃ than the mesophilic flora. In the mesophilic reactor effluent 4.6 × 10⁸ peptone-degrading colony-forming units (cfu)/ml were culturable, whereas in the thermophilic reactor effluent growth of only 5.6 × 10⁷ cfu/ml was observed. Received: 24 April 1998 / Received revision: 26 June 1998 / Accepted: 27 June 1998     

Pronounced conversion of the metal-specific activity of superoxide dismutase from Porphyromonas gingivalis by the mutation of a single amino acid (Gly155Thr) located apart from the active site

Glycine 155, which is located approximately 10 A from the active metal sites, is mostly conserved in aligned amino acid sequences of manganese-specific superoxide dismutases (Mn-SODs) and cambialistic SOD (showing the same activity with Fe and Mn) from Porphyromonas gingivalis, but is substituted for threonine in most Fe-SODs. Since Thr155 is located between Trp123 and Trp125, and Trp123 is one member of the metal-surrounding aromatic amino acids, there is a possibility that the conversion of this amino acid may cause a conversion of the metal-specific activity of cambialistic P. gingivalis SOD. To clarify this possibility, we have prepared a mutant of the P. gingivalis SOD with conversion of Gly155 to Thr. The ratios of the specific activities of Fe- to Mn-reconstituted enzyme, which are measured by the xanthine oxidase/cytochrome c method, increased from 0.6 in the wild-type to 11.2 in the mutant SODs, indicating the conversion of the metal-specific activity of the enzyme from a cambialistic type to an Fe-specific type. The visible absorption spectra of the Fe- and Mn-reconstituted mutant SODs closely resembled those of Fe-specific SOD. Furthermore, the EPR spectra of the Fe- and Mn-reconstituted mutant SODs also closely resembled those of Fe-specific SOD. Three-dimensional structures of the Fe-reconstituted wild-type SOD and Mn-reconstituted mutant SOD have been determined at 1.6 A resolution. Both structures have identical conformations, orientations of residues involved in metal binding, and hydrogen bond networks, while the side chain of Trp123 is moved further toward the metal-binding site than in wild-type SOD. A possible contribution of the structural differences to the conversion of the metal-specific activity through rearrangement of the hydrogen bond network among Trp123, Gln70, Tyr35, and the metal-coordinated solvent is discussed.     

Salmonella–Macrophage Interactions upon Manganese Supplementation

Various studies indicate the role of manganese (Mn) in the virulence of pathogens. Salmonella is an intracellular pathogen which is able to multiply within the macrophages. The present study was therefore, designed to assess the effect of Mn supplementation on Salmonella–macrophage interactions particularly in reference to Salmonella virulence and macrophage functions. A 50-fold decrease in the lethal dose 50 (LD₅₀) of Salmonella typhimurium was observed when mice were infected with Salmonella grown in the presence of Mn as compared to the LD₅₀ in the absence of Mn indicating an increase in the virulence of the organism. A significant increase was observed in the levels of superoxide dismutase (SOD) of S. typhimurium grown in presence of manganese. Upon Mn supplementation, macrophage functions were also found to be altered. Decreased phagocytic activity of macrophages interacted with Salmonella was observed in presence of Mn as compared to the activity in the absence of Mn. A significant increase was observed in the extent of lipid peroxidation along with significant decreases in the activities of SOD and catalase as well as nitrite levels of macrophages interacted with S. typhimurium upon supplementation with Mn. These observations indicate that Mn supplementation might have increased the expression of Mn transporters in Salmonella resulting in increased levels of its superoxide dismutase. The altered Salmonella function in turn might have been responsible for inhibiting phagocytosis and impairing the balance between the oxidant and antioxidant profile of macrophages, thus protecting itself by exhibiting exalted virulence.