MPO2D_PHACH
  Name and origin · Biochemical properties · Enzyme annotation · Literature · Protein features · Sequences · Cross-refs · Entry history
Name and origin
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Gene Name
Name: mpo2D
Other given name: MP-1, MnP
Protein names
Common name: manganese peroxidase
EC systematic name: Mn(II):hydrogen-peroxide oxidoreductase
Other name: peroxidase-M2, Mn-dependent (NADH-oxidizing) peroxidase
Organism
Species: Phanerochaete chrysosporium
Strain: BKM-F-1767 (ATCC 24725)
Taxonomic identifier: 5306
Taxonomic lineage: Eukaryota › Fungi › Dikarya › Basidiomycota › Agaricomycotina › Homobasidiomycetes › Corticiales › Corticiaceae › Phanerochaete
Enzyme activity is a manganese-containing, high redox-potential enzyme. In the presence of hydrogen peroxide, the manganese is oxidized from Mn(II) to Mn(III) which acts as a diffusible oxidizer degrading lignin through creation of aromatic cation radicals.
Biochemical properties
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General properties
Expression host
Substrate
Assay
Temperature Optimum(°C)
Temperature Stability(°C)
pH Optimum
pH Stability
Kinetic properties
Host Substrate Activity assay conditions Specific activity Relative activity(%) Assay Reference
Activity assay
SF-9 cells from Spodoptera frugiperda phenol red 610nm active spectrophotometric assay (phenol red oxidation).
SF-9 cells from Spodoptera frugiperda vanillylacetone 336nm active spectrophotometric assay (substrate disappearance).
SF-9 cells from Spodoptera frugiperda guaiacol 465nm active spectrophotometric assay (tetraguaiacol formation).
Pichia pastoris GS115 syringol 0.4mM manganese sulfate, 50mM sodium malonate, pH 4.5, 0.1mM hydrogen peroxide, 469nm. active spectrophotometric assay.
Pichia pastoris GS115 Mn2+ 50mM sodium malonate, pH 4.5, 0.1mM hydrogen peroxide, 270nm. active spectrophotometric assay (formation of Mn3-malonate).
native syringol 0.4mM manganese sulfate, 50mM sodium malonate, pH 4.5, 0.1mM hydrogen peroxide, 469nm. 116U/mg spectrophotometric assay.
native Mn2+ 50mM sodium malonate, pH 4.5, 0.1mM hydrogen peroxide, 270nm. active spectrophotometric assay (formation of Mn3-malonate).
Escherichia coli syringol 50mM sodium acetate, 50mM sodium succinate, pH 4.5, 3mg/ml gelatin, 0.5mM manganase sulfate, 50µM hydrogen peroxide, 470nm. active spectrophotometric assay.
Escherichia coli phenol red 50µM hydrogen peroxide, 50mM sodium-succinate, 0.1mM manganese sulfate, pH 4.5, 610nm. active spectrophotometric assay.
Escherichia coli vanillylacetone 336nm active spectrophotometric assay (substrate disappearance).
Escherichia coli guaiacol not reported. active spectrophotometric assay (disappearance of substrate).
 
Host Substrate Activity assay conditions Product formed Product analysis Reference
Product analysis
 
Host Substrate Kinetic assay conditions Km Kcat(s-1) Vmax Reference
Kinetic assay
Pichia pastoris GS115 Mn2+ 50mM sodium malonate, pH 4.5, 0.1mM hydrogen peroxide, 270nm. 0.074mM (74µM) 104
native Mn2+ 50mM sodium malonate, pH 4.5, 0.1mM hydrogen peroxide, 270nm. 0.08mM (80µM) 106
 
Other features
- All activities were Mn2+ and hydrogen peroxide dependent.
- Contains a heme prosthetic group.
- The intracellular protein may have had a lower molecular weight because it was lacking the modifications that occur during secretion.
- Activity was completely dependent on Mn2+ and hydrogen peroxide.
Enzyme annotation
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Enzyme
   Enzyme commission
1.11.1.13; Mn(II):hydrogen-peroxide oxidoreductase
Gene Ontology
GO ID Term Evidence Reference
   Biological process
GO:0055114 oxidation-reduction process IDA
GO:0042744 hydrogen peroxide catabolic process IDA
   Molecular function
GO:0016689 manganese peroxidase activity IDA
   Cellular component
GO:0005576 extracellular region IDA
Literature
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[1] Heterologous expression of active manganese peroxidase from Phanerochaete chrysosporium using the baculovirus expression system.  
Author Pease EA, Aust SD and Tien M
Journal Biochem. Biophys. Res. Commun. 1991 Sep 16;179(2):897-903.
Address Department of Molecular and Cell Biology, Pennsylvania State University University Park 16802.
Abstract The cDNA encoding Mn peroxidase isozyme H4 from Phanerochaete chrysosporium was recombined into a baculovirus and heterologously expressed in Sf9 cells. The recombinant Mn peroxidase has the same molecular weight as the native enzyme as determined by SDS-PAGE and cross-reacts with a Mn peroxidase-specific antibody. The recombinant enzyme has a slightly lower pI than the native fungal isozyme H4 indicating some differences in post-translational modification. Phenol red, guaiacol, and vanillylacetone, substrates of the native Mn peroxidase, are oxidized by the recombinant enzyme. All of the activities are dependent on both Mn (II) and H2O2.
[2] Expression of a Phanerochaete chrysosporium manganese peroxidase gene in the yeast Pichia pastoris.  
Author Gu L, Lajoie C and Kelly C
Journal Biotechnol. Prog.;19(5):1403-9.
Address L.C. Smith College of Engineering and Computer Science, Department of Chemical Engineering and Materials Science, 220 Hinds Hall, Syracuse University, Syracuse, New York 13244-1190, USA.
Abstract A gene encoding manganese peroxidase (mnp1) from Phanerochaete chrysosporium was cloned downstream of a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter in the methylotrophic yeast Pichia pastoris. Three different expression vectors were constructed: pZBMNP contains the native P. chrysosporium fungal secretion signal, palphaAMNP contains an alpha-factor secretion signal derived from Saccharomyces cerevisiae, and pZBIMNP has no secretion signal and was used for intracellular expression. Both the native fungal secretion signal sequence and alpha-factor secretion signal sequence directed the secretion of active recombinant manganese peroxidase (rMnP) from P. pastoris transformants. The majority of the rMnP produced by P. pastoris exhibited a molecular mass (55-100 kDa) considerably larger than that of the wild-type manganese peroxidase (wtMnP, 46 kDa). Deletion of the native fungal secretion signal yielded a molecular mass of 39 kDa for intracellular rMnP in P. pastoris. Treatment of the secreted rMnP with endoglycosidase H (Endo H) resulted in a considerable decrease in the mass of rMnP, indicating N-linked hyperglycosylation. Partially purified rMnP showed kinetic characteristics similar to those of wtMnP. Both enzymes also had similar pH stability profiles. Addition of exogenous Mn(II), Ca(II), and Fe(III) conferred additional thermal stability to both enzymes. However, rMnP was slightly less thermostable than wtMnP, which demonstrated an extended half-life at 55 degrees C.
[3] Expression of fungal Mn peroxidase in E. coli and refolding to yield active enzyme.  
Author Whitwam RE, Gazarian IG and Tien M
Journal Biochem. Biophys. Res. Commun. 1995 Nov 22;216(3):1013-7.
Address Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802, USA.
Abstract The cDNA encoding Mn peroxidase isozyme H4 from Phanerochaete chrysosporium was expressed in Escherichia coli. The portion of the cDNA encoding the enzyme's signal peptide, not found in the processed holoenzyme, was deleted from the cDNA. The polypeptide was produced as inactive inclusion bodies that could be solubilized in 8 M urea and the reducing agent dithiothreitol. Reconstitution of activity was accomplished by diluting the urea concentration to 2M in the presence of hemin, calcium, and oxidized glutathione. All of the additives were required for recovery of activity. The activity of the recombinant enzyme was dependent on both Mn2+ and H2O2.
[4] Manganese-dependent peroxidase from Phanerochaete chrysosporium. Primary structure deduced from cDNA sequence.  
Author Pease EA, Andrawis A and Tien M
Journal J. Biol. Chem. 1989 Aug 15;264(23):13531-5.
Address Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Abstract A cDNA clone encoding a manganese-dependent peroxidase from the filamentous fungus Phanerochaete chrysosporium was isolated and characterized. The clone, lambda MP-1, was isolated by screening a lambda gt11 expression library with polyclonal antibodies raised against a purified manganese-dependent peroxidase (isozyme H4, pI 4.5). The lambda MP-1 cDNA sequence predicts a mature protein containing 358 amino acids with a molecular weight of 37,711 preceded by a leader peptide of 24 amino acid residues. The N-terminal amino acid sequence of a purified manganese-dependent peroxidase (H4) corresponds to the sequence deduced from the cDNA. Some homology (58% in nucleotide sequence and 65% in amino acid sequence) is observed between the manganese-dependent peroxidase and lignin peroxidase isozyme H8. The highest degree of similarity is observed near the enzyme active site. Residues essential for peroxidase activity, the distal and proximal histidines, can be identified in the amino acid sequence. Near these residues, homology is also observed with several other peroxidases. Northern blot analysis of poly(A)+ RNA from nitrogen-limited P. chrysosporium cultures indicates that the level of messenger RNA correlates with expression of the enzyme and its activity. This is consistent with the regulation of the enzyme being at the level of transcription.
Protein features
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Signal Peptide (Predicted) SP(24aa)
N-Terminal (Experimental)
Structure PMID Reference
Sequence PMID Reference
CBM
Glycosylation glycosylated
Other Domains
Domain Order
CAZy family AA2
Sequences
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Length 382aa
Molecular Weight in kDa (experimental) 100 (extracellular), 55 (extracellular and N-deglycosylated), 39 (intracellular) (SDS-PAGE) , 46, 38 (SDS-PAGE)
Molecular Weight in kDa (predicted) 40.1, 38 (excluding the signal peptide)
Protein Sequence P19136
MAFGSLLAFV ALAAITRAAP TAESAVCPDG TRVTNAACCA FIPLAQDLQE TLFQGDCGED AHEVIRLTFH DAIAISQSLG PQAGGGADGS MLHFPTIEPN FSANSGIDDS VNNLLPFMQK HDTISAADLV QFAGAVALSN CPGAPRLEFM AGRPNTTIPA VEGLIPEPQD SVTKILQRFE DAGNFSPFEV VSLLASHTVA RADKVDETID AAPFDSTPFT FDTQVFLEVL LKGTGFPGSN NNTGEVMSPL PLGSGSDTGE MRLQSDFALA RDERTACFWQ SFVNEQEFMA ASFKAAMAKL AILGHSRSSL IDCSDVVPVP KPAVNKPATF PATKGPKDLD TLTCKALKFP TLTSDPGATE TLIPHCSNGG MSCPGVQFDG PA
DNA Sequence J04980
TCAGCTCTCA AGGACATCCG CACTCGAATA TCGCAATGGC CTTCGGTTCT CTCCTCGCCT TCGTGGCTCT CGCCGCCATA ACTCGCGCCG CCCCGACTGC GGAGTCTGCA GTCTGTCCAG ACGGTACCCG CGTCACCAAC GCGGCGTGCT GCGCTTTCAT TCCGCTCGCA CAGGATTTGC AAGAGACTCT GTTCCAGGGT GACTGTGGCG AAGATGCCCA CGAAGTCATC CGTCTGACCT TCCACGACGC TATTGCAATC TCCCAGAGCC TAGGTCCTCA GGCTGGCGGC GGTGCTGACG GCTCCATGCT GCACTTCCCG ACAATCGAGC CCAACTTCTC CGCCAACAGC GGCATCGATG ACTCCGTCAA CAACTTGCTT CCCTTCATGC AGAAACACGA CACCATCAGT GCCGCCGATC TTGTACAGTT CGCCGGTGCG GTCGCGCTGA GCAACTGCCC AGGTGCTCCT CGCCTCGAGT TCATGGCTGG ACGTCCGAAC ACTACCATCC CCGCAGTTGA GGGCCTCATT CCTGAGCCTC AAGACAGCGT CACCAAAATC CTGCAGCGCT TCGAGGACGC CGGCAACTTC TCGCCGTTCG AGGTCGTCTC GCTCCTGGCT TCACACACCG TTGCTCGTGC GGACAAGGTC GACGAGACCA TCGATGCTGC GCCCTTCGAC TCGACACCCT TCACCTTCGA CACCCAGGTG TTCCTCGAGG TCCTGCTCAA GGGCACAGGC TTCCCGGGCT CGAACAACAA CACCGGCGAG GTGATGTCGC CGCTCCCACT CGGCAGCGGC AGCGACACGG GCGAGATGCG CCTGCAGTCC GACTTTGCGC TCGCGCGCGA CGAGCGCACG GCGTGCTTCT GGCAGTCGTT CGTCAACGAG CAGGAGTTCA TGGCGGCGAG CTTCAAGGCC GCGATGGCGA AGCTTGCGAT CCTCGGCCAC AGCCGCAGCA GCCTCATTGA CTGCAGCGAC GTCGTCCCCG TCCCGAAGCC CGCCGTCAAC AAGCCCGCGA CGTTCCCCGC GACGAAGGGC CCCAAGGACC TCGACACGCT CACGTGCAAG GCCCTCAAGT TCCCGACGCT GACCTCTGAC CCCGGTGCTA CCGAGACCCT CATCCCCCAC TGCTCCAACG GCGGCATGTC CTGCCCTGGT GTTCAGTTCG ATGGCCCTGC CTAAACCACT CACCTCCGGC AATGCACCTT TAGTAGATGT CGATTCTATT TGGTTGATTA GAAGTACTGG GTTCTCGTGT CATATACCAC CAAGTTTTCA GAATGCAAGT CTCCTTTCTT TCCAAAAAAA AAA
Cross-references
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GenBank J04980
UniProt P19136
Protein AAA33746
RefSeq Protein
Broad
PDB
Entry history
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Entry Name MPO2D_PHACH
Previous Entry Names MnPD_PHACH
Last Modification Date 2013-04-30