Kinetic studies of peroxiredoxin 6 from Arenicola marina: Rapid oxidation by 3 hydrogen peroxide and peroxynitrite but lack of reduction by hydrogen sulfide

, respectively, at pH 7.4 and 25°C. Reduction of tert-butyl hydroperoxide was slower. 34 The pK a of the peroxidatic thiol of AmPrx6 was determined as 5

Cloning and characterization of Arenicola marina peroxiredoxin6

Peroxiredoxins (PRDXs) are a superfamily of thiol-dependent selenium- and heme-free peroxidases found in all phyla. PRDXs are mechanistically divided into three subfamilies namely typical 2-Cys, atypical 2-Cys and 1-Cys PRDXs. To reduce peroxides, the N-terminal conserved peroxidatic Cys is first oxidized into sulfenic acid. According to the subfamily, this intermediate is reduced by forming a disulfide bond either with a resolving Cys of another monomeric entity (typical 2-Cys) or of the same molecule (atypical 2-Cys). Finally, in 1-Cys PRDXs, the sulfenic acid is reduced by a heterologous thiol-containing reductant. In the search of the homolog of human 1-Cys PRDX6 in Arenicola marina (Am), an annelid worm living in the sulfide-rich sediments of the intertidal zones, we have cloned and characterized a novel PRDX exhibiting high sequence homology with its mammalian counterpart. However, AmPRDX6, possesses five Cys among which one is at a homologous position to the resolving Cys of typical 2-Cys PRDXs. Here, we report that AmPRDX6 has a catalytic mechanism involving intermolecular disulfide bonds. The role of these Cys and their interactions have been investigated. We report also the kinetic characterization of the enzyme which shows that AmPRDX6 reacts with hydrogen peroxide and peroxynitrite with high rate constants. This fast reactivity can be, to some extent, explained by the low pKa of catalytic thiol. The recovery step was also examined using many potential physiological reductants but none were able to significantly reduce the enzyme. Moreover, to gain insights into the cellular functions of AmPRDX6, CHO cells were used as an in vitro model in which the enzyme was overexpressed. Cells were exposed to an acute oxidative stress but AmPRDX6 did not confer cytoprotection. In conclusion, this work shows that AmPRDX6 belongs to a transient group exhibiting sequence homologies with mammalian 1-Cys PRDX6 but must be mechanistically classified into typical 2-Cys PRDXs.(BIOL 3) -- UCL, 201

Kinetic studies of peroxiredoxin 6 from Arenicola marina: Rapid oxidation by hydrogen peroxide and peroxynitrite but lack of reduction by hydrogen sulfide

Arenicola marina lives in marine environments where hydrogen peroxide concentrations reach micromolar levels. The annelid also forms reactive species through metabolic pathways. Its antioxidant systems include a cytosolic peroxiredoxin, peroxiredoxin 6 (AmPrx6 or AmPRDX6) that shows high homology to the mammalian 1-Cys peroxiredoxin. Previous work confirmed the peroxidase activity of AmPrx6 in the presence of dithiotreitol. Herein, we performed an in vitro kinetic characterization of the recombinant enzyme. AmPrx6 reduced hydrogen peroxide and peroxynitrite with rate constants of 1.1 × 107 and 2 × 106 M−1 s−1, respectively, at pH 7.4 and 25 °C. Reduction of tert-butyl hydroperoxide was slower. The pKa of the peroxidatic thiol of AmPrx6 was determined as 5.1 ± 0.2, indicating that it exists as thiolate, the reactive species, at physiological pH. The reductive part of the catalytic cycle was also explored. Hydrogen sulfide, present in millimolar concentrations in marine sediments where the annelid lives and that is able to reduce the mammalian 1-Cys peroxiredoxin, did not support AmPrx6 peroxidase activity. The enzyme was not reduced by other potential physiological reductants tested. Our data indicate that in this annelid, Prx6 could contribute to peroxide detoxification in the presence of a so far unidentified reducing counterpart

Cloning and characterization of Arenicola marina peroxiredoxin 6, an annelid two-cysteine peroxiredoxin highly homologous to mammalian one-cysteine peroxiredoxins.

Peroxiredoxins (PRDXs) are a superfamily of thiol-dependent peroxidases found in all phyla. PRDXs are mechanistically divided into three subfamilies, namely typical 2-Cys, atypical 2-Cys, and 1-Cys PRDXs. To reduce peroxides, the N-terminal peroxidatic Cys of PRDXs is first oxidized into sulfenic acid. This intermediate is reduced by forming a disulfide bond either with a resolving Cys of another monomeric entity (typical 2-Cys) or of the same molecule (atypical 2-Cys). In 1-Cys PRDXs, the resolving Cys is missing and the sulfenic acid of the peroxidatic Cys is reduced by a heterologous thiol-containing reductant. In search of a homolog of human 1-Cys PRDX6 in Arenicola marina, an annelid worm living in intertidal sediments, we have cloned and characterized a PRDX exhibiting high sequence homology with its mammalian counterpart. However, A. marina PRDX6 possesses five Cys among which two Cys function as peroxidatic and resolving Cys of typical 2-Cys PRDXs. Thus, A. marina PRDX6 belongs to a transient group exhibiting sequence homologies with mammalian 1-Cys PRDX6 but must be mechanistically classified into typical 2-Cys PRDXs. Moreover, PRDX6 is highly expressed in tissues directly exposed to the external environment, suggesting that this PRDX may be of particular importance for protection against exogenous oxidative attacks

The crystal structure of the C45S mutant of annelid Arenicola marina peroxiredoxin 6 supports its assignment to the mechanistic typical 2-Cys subfamily without any formation of toroid-shaped decamers

The peroxiredoxins (PRDXs) define a superfamily of thiol-dependent peroxidases able to reduce hydrogen peroxide, alkyl hydroperoxides, and peroxynitrite. Besides their cytoprotective antioxidant function, PRDXs have been implicated in redox signaling and chaperone activity, the latter depending on the formation of decameric high-molecular-weight structures. PRDXs have been mechanistically divided into three major subfamilies, namely typical 2-Cys, atypical 2-Cys, and 1-Cys PRDXs, based on the number and position of cysteines involved in the catalysis. We report the structure of the C45S mutant of annelid worm Arenicola marina PRDX6 in three different crystal forms determined at 1.6, 2.0, and 2.4 A resolution. Although A. marina PRDX6 was cloned during the search of annelid homologs of mammalian 1-Cys PRDX6s, the crystal structures support its assignment to the mechanistically typical 2-Cys PRDX subfamily. The protein is composed of two distinct domains: a C-terminal domain and an N-terminal domain exhibiting a thioredoxin fold. The subunits are associated in dimers compatible with the formation of intersubunit disulfide bonds between the peroxidatic and the resolving cysteine residues in the wild-type enzyme. The packing of two crystal forms is very similar, with pairs of dimers associated as tetramers. The toroid-shaped decamers formed by dimer association and observed in most typical 2-Cys PRDXs is not present. Thus, A. marina PRDX6 presents structural features of typical 2-Cys PRDXs without any formation of toroid-shaped decamers, suggesting that it should function more like a cytoprotective antioxidant enzyme or a modulator of peroxide-dependent cell signaling rather than a molecular chaperone