1887

Abstract

A novel ligninolytic peroxidase gene () was cloned and characterized from a poroid brown-rot fungus, . The genomic DNA of the fungus harboured two copies of , with a length of 2111 bp, interlaced with 12 introns, while the full-length cDNA was 1183 bp, with a 66 bp signal peptide and an ORF of 990 bp. The three-dimensional molecular structure model was comparable to that of the versatile peroxidase of . was cloned into vector pQE31, successfully expressed in strain M15 under the control of the T5 promoter and produced a non-glycosylated protein of about 38 kDa, pI 5.42. The native and recombinant ACLnP was capable of oxidizing the redox mediator veratryl alcohol, and also decolorized bromophenol blue and 2,6-dimethoxyphenol dyes, implicating a functional extracellular peroxidase activity. The significance of discovering a functional gene in in terms of wood degradation and colonization capacity in its unique niche is discussed.

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2009-02-01
2020-01-22
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Map of expression vector pQE31- , containing a T5 promoter and an inserted 6xHis-tag.

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Comparison of amino acid sequences of ACLnP with extracellular peroxidases of other wood-rot basidiomycetes: PCLIG1 (Q01775), PCLIG2 (AAA33735.1), PCLIGA (CAA38177.1), PCLIGH8 (P06181.1), PCLIGH2 (P11542.2), PCLIGJ (AAD46494.1), PCLIGH5 (AAA33734.1), PCLIG3 (CAA35939.1), PCMnP (AAA33743.1), PCMnP1 (AAA33742.1), PCMnP2 (AAB30859.1), PCMnP3 (AAA62243.1); TVLPGIV (CAA83228.1), TVLPG1 (AAA34049.1), TVLPG3 (CAA83146.1), TVLPG7 (CAA83147.1), TVLPGV (CAA54398.1), TVMNP2 (CAA83148.1); PEVPS1 (Q9UVP6.1), PEVP (AAZ04666.1), PEVPL1 (Q9UR19.1), PEVPL2 (O94753.1); CdP (AAZ14938.1); CmP (S78602); CcP1 (CAA49216.1); CcP2 (1H3J); P (BAA09861.1); AbMnP (CAG27835.1); GaaMnP (BAA88392.1); GauMnP (ABB77244.1); GfMnP (ABB77243.1); TCLIG (BAE46585.1); ArHP (CAA00083.1); PP (EAA53513.1).

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Nucleotide and translated amino acid sequences of . Introns are numbered I-XII. Inverted triangle, start of transcript; asterisk, stop codon; green dotted underline, transmembrane protein helix structure; purple arrow, signal peptide cleavage site; star, proximal haem ligand and distal histidines; diamond, three residues adjacent to the proximal histidines; green letters, possible glycosylation sites. The four TATA boxes and three CAAT elements are shown in blue; putative regulatory elements are indicated by underlining (two SP1 and HSF, one AP1, AP2, RFX2, C/EBP and GATA binding factor: SP1, stimulating protein 1; HSF, heat-shock factor; AP1, factor involved in response to oxidative stress/oxygen detoxification and metal resistance; AP2, activator protein 2; RFX2, enhancer factor; C/EBP, enhancer binding protein). The 5′ upstream sequences of were predicted by using MatInspector ( www.genomatix.de) and AliBaba 2.1 ( www.gene-regulation.com) software.

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Comparison of the number, position and size of the introns (orange blocks) of with genes of LiPs, MnPs and VPs from other white-rot basidiomycetes. The distance between vertical lines represents 500 nt. LiPs: is from ACT1; PCLiG1 (M24082.1), PCLiG2 (M92644.1), PCLiGA (X54257.1), PCLiGH2 (X15599.1), PCLiGJ (AF140062.1) and PCLiGH5 (X55343.1) are from ; TVLiG1 (M64993.1), TVLiG3 (Z30666.1), TVLiG5 (D86493.1) and TVLiG7 (Z30667.1) are from . MnPs: PCMNP1 (M77513.1), PCMNP2 (S69963.1) and PCMNP3 (U70998.1) are from ; STVMNP2 (Z30668.1) is from . VPs: PEVP (DQ056374.1), PEVPL1 (AF007223.1), PEVPL2 (AF007224.1) and PEVPS1 (AF175710.1) are from .

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Analysis of LiP, BG, EBG and 18S RNA from the mycelium (blue bars) or fruiting body (orange bars) of by Q-PCR.

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Alignment and comparison of from five fosmid clones [b16 ( ), g20, k16, k17, p11]. With the exception of clone g20, with 8 nucleotide substitutions, the sequences in the other four fosmid clones are identical.

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Molecular model of ACLiP, consisting of 13 α-helices, 3 β-sheets, 2 Ca binding sites, an internal haem functional block, and N and C termini, predicted based on the available crystallographic structures of LiP, MnP, VP and peroxidase from other white-rot basidiomycetes. Stereo view of the partial molecular structure of ACLnP. The internal haem functional block is surrounded by conserved amino acids. The possible short- and long-range electron transfer routes between amino acids, or between amino acids and haem (dotted line) were predicted using PyMOL software ( http://pymol.sourceforge.net).

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Molecular model of ACLiP, consisting of 13 α-helices, 3 β-sheets, 2 Ca binding sites, an internal haem functional block, and N and C termini, predicted based on the available crystallographic structures of LiP, MnP, VP and peroxidase from other white-rot basidiomycetes. Stereo view of the partial molecular structure of ACLnP. The internal haem functional block is surrounded by conserved amino acids. The possible short- and long-range electron transfer routes between amino acids, or between amino acids and haem (dotted line) were predicted using PyMOL software ( http://pymol.sourceforge.net).

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Superimposition of 3-D molecular structures of ACLnP (ACLiP, orange) with Phanerochaete chrysosporium LiP (PCLiP, green) and MnP (PCMnP, purple), VP (PEVP, blue) and peroxidase (ArP, pale blue) retrieved from the PDB database ( www.rcsb.org/pdb/home/home.do). Calcium (Ca ), pale green; manganese (Mn ), pale grey.

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The gene containing 12 introns. The 770 bp at the 3′ end without RV, I and RI restriction sites was used as the gene-specific probe for Southern blot analyses.

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Alignment of ACLnP (ACLiP, clone g20) with the VP precursor VPL1 sequence from the NCBI database.

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Primers used for PCR and Q-PCR

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Properties of and the prediction of regulatory elements in the promoter region

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