What is the function of peroxidase

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Title:Biochemical characterization and bioinformatic sequence analysis of plasma membrane-bound peroxidases from maize roots (Zea mays L.)Other titles:Biochemical characterization and sequence analysis of plasma membrane-bound peroxidases isolated from corn roots (Zea mays L.)Language:GermanAuthor:Mika, AngelaTags:Biochemical characterization; Rboh; peroxidase; plasma membrane; membrane-bound; sequence analysis; biochemical characterizationGND keywords:Peroxidase; Plasma membrane; Membrane proteins; Sequence analysis Publication date:2005Day of the oral exam:2005-12-09Summary:
In the last two decades there have been several indications for the existence of a plasma membrane-bound peroxidase activity in plant cells. For this membrane-bound enzyme activity, among other things, participation in the production of active oxygen species (AOS) and a detoxification of AOS to protect the membrane in connection with pathogen defense and cell wall structure were discussed. However, contamination by soluble apoplastic or cytosolic proteins could not be excluded for these investigations. In the meantime, however, evidence of the existence of peroxidase activities firmly bound to the membrane has been provided (Mika, 2001). In the present work, these membrane-bound peroxidase activities from plasma membranes of maize roots (Zea mays L.) were partially purified and biochemically characterized for the first time. The sequences of the associated enzymes were identified and analyzed using bioinformatics.

Four different plasma membrane bound peroxidases (pmPOX1, pmPOX2a, pmPOX2b and pmPOX3) could be detected. Non-covalently bound heme groups were identified as prosthetic groups of the proteins. The native molecular masses of the enzymes were determined by gel filtration in comparison with standard proteins. Analyzes of the partially purified proteins by a modified non-reducing SDS-PAGE in combination with a heme staining confirmed the masses derived from the chromatographic purification (138, 98, 57 and 55 kDa). A glycosylation of the proteins could be shown by different methods. Both anionic and neutral and cationic values ​​appeared as isoelectric points.

The partially purified peroxidase activities showed the broad substrate specificity characteristic of these enzymes. Since the enzyme activities remained unchanged in the presence of 4-hydroxymercuric benzoic acid and the enzymes showed high affinities for phenolic substrates, the pmPOX investigated could be classified in class III of the superfamily of peroxidases from plants, bacteria and fungi according to the classification according to Welinder (1992) classify.
The formation of AOS is essential for many peroxidase-mediated functions. In connection with various stress factors, essentially two possible sources for the massive production of AOS (oxidative burst) are discussed: 1) apoplastic peroxidases and 2) a diphenylene iodonium sensitive NAD (P) H oxidase (respiratory burst oxidase homolog, Rboh). In order to clarify whether and under what conditions the plasma membrane-bound peroxidases can produce AOS in vitro, their NAD (P) H oxidase activities were investigated in detail. For example, pmPOX1, pmPOX2a and pmPOX2b were able to produce large amounts of AOS in vitro. However, some characteristics of these activities, in particular a pH optimum in the acidic pH range as well as a lack of stimulability by calcium, showed clear differences from the properties previously postulated for the participation of peroxidases in the oxidative burst.

By mass spectrometric analysis of the proteins and in silico cloning, the full length nucleic acid sequences of pmPOX1 and pmPOX2b could be identified and three possible sequences for pmPOX3 could be determined. In a bioinformatic analysis of these sequences, numerous other characteristics of the enzymes could be derived and compared with the experimentally determined data. Structural aspects of the corresponding genes were derived on the basis of genomic nucleic acid sequences of the pmPOX.
At the amino acid level, all pmPOX sequences showed the greatest sequence matches with peroxidases of class III and their classic characteristics: In addition to conserved peroxidase domains, it was possible to identify, among other things, putative disulfide bridges, calcium binding sites, glycosylation sites and N-terminal signal peptides that direct the ER. The three-dimensional structure of pmPOX1 and pmPOX2b could be modeled using similar, soluble class III peroxidases. Bioinformatic predictions of hydrophobic domains in comparison with solubilization experiments indicate that the signal peptide directing to the ER is not split off and the proteins are anchored by an N-terminal transmembrane domain.

Homologous proteins of the plasma membrane-bound peroxidases could be determined in different monocotyledon and dicotyledon plant species and the individual pmPOX in the form of Passardi et al. (2004) established subgroups for secretory peroxidases. In silico Northern blot analysis suggests that both pmPOX1 and pmPOX2b are mainly expressed in root tissues. Based on these results, possible functions of the plasma membrane-bound enzymes were discussed.

In the last decades several investigations indicated the presence of a plasma membrane-bound peroxidase activity in plant cells. For these enzymes located directly at the membrane several functions were postulated including protection of the membrane or production of active oxygen species (AOS) in pathogen defense or cell wall formation. This study is the first to purify and characterize the responsible enzymes from highly enriched and thoroughly washed plasma membranes. The sequences of the corresponding enzymes were identified and analyzed.

Four different obviously tightly plasma membrane-bound peroxidases (pmPOX) could be partially purified (138, 98, 57, and 55 kDa in non-reducing SDS-PAGE). Non-covalent bound heme groups were identified as prosthetic groups. The glycosylation of the proteins could be shown by different methods. The determination of isoelectric points showed anionic as well as neutral and cationic values.

The H2O2-detoxifying activities of the partially purified pmPOXs showed distinct properties in dependence on substrates and effectors. Since the enzymes were localized at the plasma membrane, not effected by p-chloromercuribenzoate, and showed high affinities to phenolic compounds, they could be categorized as class III peroxidases of the superfamily of peroxidases from plants, fungi and bacteria according to Welinder et al. (1992).

Besides H2O2 detoxification the formation of AOS is essential for many peroxidase-dependent functions. So far two main sources for the massive production of AOS, the so-called oxidative burst, in presence of several abiotic or biotic stress factors have been discussed: 1) apoplastic peroxidases and 2) a diphenylene iodonium-sensitive
NAD (P) H oxidase (respiratory burst oxidase homologue, Rboh). In order to clear whether and how much the plasma membrane-bound peroxidases are able to participate in vitro in AOS production, their NAD (P) H oxidase activities were extensively characterized.

The full length nucleotide sequences of pmPOX1 and pmPOX2b could be identified by nanoelectrospray mass spectrometry analysis and in silico cloning of the purified proteins. Additionally, three possible sequences for pmPOX3 could be found. Relative and theoretical molecular masses and isoelectric points were compared. Several other data were deduced from the sequences, e.g. high sequence similarity to other class III peroxidases, conserved and variable regions, homologues, and putative protein structures. Bioinformatic predictions of hydrophobic domains in comparison with solubilization experiments suggest a non-cleavable ER signal peptide for each protein, and an intrinsic binding of the pmPOXs with an N-terminal transmembrane domain. Derived from in silico northern blot analysis pmPOX1, pmPOX2b and pmPOX3 seem to be expressed mainly in roots. Possible functions of the plasma membrane-bound peroxidases were discussed.
Url:https://ediss.sub.uni-hamburg.de/handle/ediss/1210URN:urn: nbn: de: gbv: 18-27694Document type:dissertationSupervisor:Böttger, Michael (Prof. Dr.)Included in the collections:Electronic dissertations and habilitations