Bacterial Peroxidase Research Articles

Superfamily of plant, fungal and bacterial peroxidases

New peroxidase structures have significantly increased our understanding of the evolutionary and functional relationships within the plant peroxidase superfamily. Three distantly related structural classes have emerged: mitochondrial yeast cytochrome c peroxidase, chloroplast and cytosol ascorbate peroxidases, and gene duplicated bacterial peroxidase (class I); secretory fungal peroxidases (class II); and, classical, secretory plant peroxidases (class III).

Bacterial catalase-peroxidases are gene duplicated members of the plant peroxidase superfamily

Bacterial catalase-peroxidases are enzymes containing 0.5-1.0 heme per subunit. The identical subunits are generally 80 kDa in size, and the sequenced subunits of E. coli, S. typhimurium and B. stearothermophilus contain 726-731 amino acid residues per subunit. The heme-containing peroxidases of plants, fungi and yeast are monomeric, homologous and 290-350 residues in size. Analyses of the amino acid sequences indicate that the double length of the bacterial peroxidases can be ascribed to gene duplication. Each half is homologous to eukaryotic, monomeric peroxidase and can be modelled into the high-resolution crystal structure of yeast cytochrome c peroxidase. The comparisons and modelling have predicted: (1) the C-terminal half does not bind heme, and bacterial peroxidases have one heme per subunit; (2) the ten dominating helices observed in the yeast enzyme are highly conserved and connected by surface loops which are often longer in the bacterial peroxidases; and (3) yeast cytochrome c peroxidase has evolved more slowly than other known peroxidases. The study has revealed ten invariant residues and a number of highly conserved residues present in peroxidases of the plant peroxidase superfamily and provides a basis for rationally engineered peroxidases.

Effects of bacterial lignin peroxidase on organic carbon mineralization in soil, using recombinant Streptomyces strains

To study the effects of bacterial lignin peroxidase ALip-P3 of Streptomyces viridosporus T7A on the rate of organic carbon turnover in soil, purified lignin peroxidase, with and without addition of H2O2, was added to sterile and nonsterile silt loam soil. Recombinant Streptomyces lividans strains expressing plasmid-encoded ALip-P3 were also inoculated into the soil. Carbon mineralization was monitored by measuring the rate of CO2 evolution from the soil. In sterile soil, lignin peroxidase addition altered carbon turnover, by increasing the CO2 evolution rate above the near-zero rate of sterile, uninoculated soil. H2O2, when added alone, had no effect, and its addition in combination with peroxidase gave results similar to peroxidase alone. This effect was also observed upon addition of lignin peroxidase to sterile soil already inoculated with S. viridosporus T7A. The increases in soil CO2 evolution rates were also observed in experiments using nonsterile soil. However, results showed more variation, and the effect was shorter lived as a result of lessened peroxidase stability. Three recombinant S. lividans strains expressing the ALip-P3 gene in plasmid pIJ702.LP were also inoculated into soil. There were no significant differences in CO2 evolution rates for sterile soil inoculated with recombinants as compared with sterile soil inoculated with wild-type S. lividans strains. However, in nonsterile soil, addition of the recombinants caused a significantly greater increase in the CO2 evolution rate as compared with the corresponding wild types or S. viridosporus T7A. The effect was short lived, lasting about 5 days. Both the recombinant and wild-type Streptomyces survived in the soil for at least 30 days, and pIJ702.LP was stable in the recombinants in soil. Plasmid pIJ702.LP was transformed into three mutants of S. viridosporus T7A that lacked lignin peroxidase. Plasmid-expressing transformants regained the ability to produce lignin peroxidase. The results show that addition of lignin peroxidase ALip-P3 to soil transiently enhanced the short-term rate of carbon mineralization in the soil. The enhancement was lignin peroxidase specific, since substitution of horseradish peroxidase for lignin peroxidase in the soil addition studies resulted in no enhancement of CO2 evolution. In addition, pIJ702.LP-expressing S. lividans strains also caused the effect, which was significant only in nonsterile soil. Thus, lignin peroxidase ALip-P3 appears to affect the short-term turnover rate of lignin-derived organic carbon in soil, and normal, low lignin peroxidase concentrations in soil may limit the initial turnover rate of lignified plant residues in soil. This is the first report of a genetically engineered microorganism having a measurable effect on a biogeochemical cycle in soil. Key words: Streptomyces, recombinant, lignin, peroxidase, soil.

Evaluation of Hematuria in Children and Adolescents

Hematuria is a frequent abnormal clinical finding in the genitourinary system, second only to urinary tract infection. The pediatrician needs to have an appropriate and organized approach to the examination of children with hematuria, to identify common causes or diseases and to identify those who need a more extensive evaluation of their problem and those who need urologic evaluation. The prevalence of microscopic hematuria among school-aged children ranges from 0.4% to 2.1%. Despite this frequency, it would appear that the presence of hematuria alone is rarely indicative of serious illness. DEFINITIONS The literature concerning hematuria is inconsistent in the various methods used to detect it and in the differing criteria for diagnosing hematuria. The most sensitive test for detecting the presence of blood in the urine is a reagent strip impregnated with orthotolodine-peroxide and enhanced with 6-methoxyquinolone. The commonly used dipsticks (Chemstrip-Boehringer Mannheim; Multistix or Labstix, Ames Division of Miles Laboratories) are sensitive. In the presence of hemoglobin (or another oxidizing agent), a blue coloration of the strip occurs. False-negative results are unusual but have been reported in the presence of high urinary concentrations of ascorbic acid. False-positive results (also unusual) are occasionally demonstrated in heavily infected urine where bacterial peroxidase is being liberated in high quantity.

Evaluation of hematuria in children and adolescents.

Hematuria is a frequent abnormal clinical finding in the genitourinary system, second only to urinary tract infection. The pediatrician needs to have an appropriate and organized approach to the examination of children with hematuria, to identify common causes or diseases and to identify those who need a more extensive evaluation of their problem and those who need urologic evaluation. The prevalence of microscopic hematuria among school-aged children ranges from 0.4% to 2.1%. Despite this frequency, it would appear that the presence of hematuria alone is rarely indicative of serious illness. DEFINITIONS The literature concerning hematuria is inconsistent in the various methods used to detect it and in the differing criteria for diagnosing hematuria. The most sensitive test for detecting the presence of blood in the urine is a reagent strip impregnated with orthotolodine-peroxide and enhanced with 6-methoxyquinolone. The commonly used dipsticks (Chemstrip-Boehringer Mannheim; Multistix or Labstix, Ames Division of Miles Laboratories) are sensitive. In the presence of hemoglobin (or another oxidizing agent), a blue coloration of the strip occurs. False-negative results are unusual but have been reported in the presence of high urinary concentrations of ascorbic acid. False-positive results (also unusual) are occasionally demonstrated in heavily infected urine where bacterial peroxidase is being liberated in high quantity.

An Alkyl Hydroperoxide Reductase from Salmonella typhimurium Involved in the Defense of DNA against Oxidative Damage: Purification and properties

A peroxide reductase (peroxidase) which converts lipid hydroperoxides and other alkyl hydroperoxides to the corresponding alcohols, using either NADH or NADPH as the reducing agent, has been identified in both Salmonella typhimurium and Escherichia coli. This enzyme is shown to play a role in protecting against alkyl hydroperoxide mutagenesis. To our knowledge this work represents the first description of an NAD(P)H peroxidase in enteric bacteria and the first reported bacterial peroxidase to exhibit high activity toward alkyl hydroperoxides. A high performance liquid chromatography-based assay for the alkyl hydroperoxide reductase has been developed by monitoring the reduction of cumene hydroperoxide, a model alkyl hydroperoxide. By using this assay, the enzyme has been purified from a S. typhimurium regulatory mutant, oxyR1, which overexpresses a number of proteins involved in defenses against oxidative damage, and which contains 20-fold more of the alkyl hydroperoxide reductase than the wild-type strain. The purified activity requires the presence of two separable components having subunit molecular weights of 22,000 and 57,000. The 57-kDa protein contains a bound FAD cofactor and can use either NADH or NADPH as an electron donor for the direct reduction of redox dyes, or of alkyl hydroperoxides when combined with the 22-kDa protein. This enzyme may thus serve as a prokaryotic equivalent to the glutathione reductase/glutathione peroxidase system in eukaryotes.

Whole-cell bacterial peroxidase test with isoproterenol as the hydrogen donor

The β-adrenergic compound isoproterenol was used as oxidizable reagent in a whole-cell assay for the detection of bacterial peroxidase activities. Isoproterenol has been shown to constitute a useful reagent for detecting peroxidase activities in enzymatic tests, utilizing standard purified enzymes, and in the microbiological application proposed. The procedure developed is simple and rapid to perform. In contrast to currently used whole-cell tests for bacterial peroxidases, the assay described here does not need preliminary permeabilization; moreover, the compound utilized does not have related toxicological problems. Therefore, the isoproterenol assay may represent a low-cost safe additional peroxidase test in clinical bacteriology.

Precision resistance measurements : E. H. Rayner. ( The Physical Society of London, Proceedings, vol. xxvii, part iv.)

Neisseria gonorrhoeae is an obligate human pathogen that expresses an array of molecular systems to detoxify reactive oxygen species as defense mechanisms during colonization and infection. One of these is the bacterial peroxidase that reduces H2O2 to water in its periplasm. The soluble form of this enzyme was heterologously expressed in E. coli in the holo-form binding two c-types hemes, a high-potential E heme and a low-potential P heme, with redox potentials of (+ 310 mV) and (− 190 mV/− 300 mV), respectively in the presence of calcium ions, at pH 7.5. Visible and EPR spectroscopic analysis together with activity assays indicate the presence of a calcium dependent reductive activation mechanism in thgonorrhoeaeNeisseria gonorrhoeae bacterial peroxidase, in which P heme is bis-His coordinated low-spin in the fully oxidized state of the enzyme, and becomes penta-coordinated high-spin upon reduction of E heme in the presence of calcium ions. The activated enzyme has a high affinity for H2O2 (KM of 4 ± 1 μM), with maximum activity being attained at pH 7.0 and 37 °C, with the rate-limiting step in the catalytic cycle being the electron transfer between the two hemes. In this enzyme, dimer formation is not promoted at high ionic strength, thus differing from the classical bacterial peroxidases. These results contribute to the understanding of the involvement of Neisseria gonorrhoeae bacterial peroxidase has a first line defense mechanism against exogenously produced hydrogen peroxide in the host environment.