Michael H. Gold

In vitro depolymerization of lignin by manganese peroxidase of Phanerochaete chrysosporium

Homogeneous manganese peroxidase catalyzed the in vitro partial depolymerization of four different 14C-labeled synthetic lignin preparations. Gel permeation profiles demonstrated significant depolymerization of 14C-sidechain-labeled syringyl lignin, a 14C-sidechain-labeled syringyl-guaiacyl copolymer (angiosperm lignin), and depolymerization of 14C-sidechain- and 14C-ring-labeled guaiacyl lignins (gymnosperm lignin). 3,5-Dimethoxy-1,4-benzo-quinone, 3,5-dimethoxy-1,4-hydroquinone, and syringylaldehyde were identified as degradation products of the syringyl and syringyl-guaiacyl lignins. These results suggest that manganese peroxidase plays a significant role in the depolymerization of lignin by Phanerochaete chrysosporium.

Degradation of pentachlorophenol by Phanerochaete chrysosporium: intermediates and reactions involved.

Under nitrogen-limiting, secondary metabolic conditions, the lignin-degrading basidiomycete Phanerochaete chrysosporium rapidly degrades pentachlorophenol. The pathway for the degradation of pentachlorophenol has been elucidated by the characterization of fungal metabolites and oxidation products generated by purified lignin peroxidase (LiP) and manganese peroxidase (MnP). The multi-step pathway is initiated by a LiP- or MnP-catalysed oxidative dechlorination reaction to produce tetrachloro-1,4-benzoquinone. Under primary or secondary metabolic conditions, the quinone is further degraded by two parallel pathways with cross-links. The quinone is reduced to tetrachlorodihydroxybenzene, which can undergo four successive reductive dechlorinations to produce 1,4-hydroquinone, and the latter is o-hydroxylated to form the final aromatic metabolite, 1,2,4-trihydroxybenzene. Alternatively, the tetrachloro-1,4-benzoquinone is converted, either enzymically or nonenzymically, to 2,3,5-trichlorotrihydroxybenzene, which undergoes successive reductive dechlorinations to produce 1,2,4-trihydroxybenzene. Finally, at several points, hydroxylation reactions convert chlorinated dihydroxybenzenes to chlorinated trihydroxybenzenes, linking the two pathways at each of these steps. Presumably, the 1,2,4-trihydroxybenzene produced in each pathway is ring-cleaved with subsequent degradation to CO2. In contrast to the oxidative dechlorination step, the reductive dechlorinations and hydroxylations occur during both primary and secondary metabolic growth. Apparently, all five chlorine atoms are removed from the substrate prior to ring cleavage.

Use of polymeric dyes in lignin biodegradation assays

Publisher Summary Various 14 C-radiolabeled and unlabeled substrates have been used to screen for ligninolytic activity. However, these assays are relatively slow and cumbersome and often require synthesis of substrates that are not commercially available. The polymeric dyes used in these assays are inexpensive and can be obtained commercially in high purity. They are stable and readily soluble, have high extinction coefficients, and low toxicity toward Phanerochaete chrysosporium and other white rot fungi and bacteria tested, o-Anisidin and other low-molecular-weight dyes that have been used in similar assays could be taken into the cells, whereas polymeric dyes will remain extracellular, at least during the initial stages of degradation, and thus will provide a better model for lignin degradation. A growing body of evidence indicates that the dyes serve as substrates for at least some component(s) of the lignin degradative system and that dye decolorization is correlated with the onset of secondary metabolism and ligninolytic activity. Recent studies indicate that only lignin-degrading fungi are able to decolorize the dye Poly B-411 and that efficiency of decolorization is correlated with the ability to degrade several lignin model compounds.

The green fluorescent protein gene functions as a reporter of gene expression in Phanerochaete chrysosporium

ABSTRACT The enhanced green fluorescent protein (GFP) gene (egfp) was used as a reporter of gene expression driven by the glyceraldehyde-p-dehydrogenase (gpd) gene promoter and the manganese peroxidase isozyme 1 (mnp1) gene promoter in Phanerochaete chrysosporium. Four different constructs were prepared. pUGGM3′ and pUGiGM3′ contain the P. chrysosporium gpd promoter fused upstream of the egfpcoding region, and pUMGM3′ and pUMiGM3′ contain the P. chrysosporium mnp1 promoter fused upstream of theegfp gene. In all constructs, the egfp gene was followed by the mnp1 gene 3′ untranslated region. In pUGGM3′ and pUMGM3′, the promoters were fused directly withegfp, whereas in pUGiGM3′ and pUMiGM3′, following the promoters, the first exon (6 bp), the first intron (55 bp), and part of the second exon (9 bp) of the gpd gene were inserted at the 5′ end of the egfp gene. All constructs were ligated into a plasmid containing the ura1 gene of Schizophyllum commune as a selectable marker and were used to transform a Ural1 auxotrophic strain of P. chrysosporium to prototrophy. Crude cell extracts were examined for GFP fluorescence, and where appropriate, the extracellular fluid was examined for MnP activity. The transformants containing a construct with an intron 5′ of theegfp gene (pUGiGM3′ and pUMiGM3′) exhibited maximal fluorescence under the appropriate conditions. The transformants containing constructs with no introns exhibited minimal or no fluorescence. Northern (RNA) blots indicated that the insertion of a 5′ intron resulted in more egfp RNA than was found in transformants carrying an intronless egfp. These results suggest that the presence of a 5′ intron affects the expression of theegfp gene in P. chrysosporium. The expression of GFP in the transformants carrying pUMiGM3′ paralled the expression of endogenous mnp with respect to nitrogen and Mn levels, suggesting that this construct will be useful in studyingcis-acting elements in the mnp1 gene promoter.

Manganese peroxidase from Phanerochaete chrysosporium

Publisher Summary Manganese peroxidase is an extracellular enzyme expressed during secondary metabolism as part of the lignin-degradative system of Phanerochaete chrysosporium . This chapter discusses purification procedure of manganese peroxidase. Manganese peroxidase can be isolated from any wild-type strain of P. chrysosporium. The chapter also discusses the properties of the enzyme. In the presence of H 2 O 2 the enzyme oxidizes polymeric and other dyes, lignin model compounds, and various phenols. Mn(III) is capable of oxidizing all of the organic substrates that are oxidized by the enzyme system. In the absence of exogenous H 2 O 2 , the enzyme also acts as an NAD(P)H oxidase generating H 2 O 2 . This reaction can be coupled to the oxidation of ABTS in the absence of exogenous H 2 O 2 , suggesting that the manganese peroxidase may play a role in H 2 O 2 production by the fungus under ligninolytic conditions.

Characterization of the gene encoding manganese peroxidase isozyme 3 from Phanerochaete chrysosporium.

The gene encoding manganese peroxidase isozyme 3 (MnP3) from the white-rot basidiomycete Phanerochaete chrysosporium was cloned and sequenced. The mnp3 gene encodes a mature protein of 357 amino acids with a 25 amino-acid signal peptide. The amino acids involved in peroxidase function, as well as those forming the MnII binding site and those involved in disulfide bond formation, are conserved in the MnP3 sequence. The mnp3 gene has six introns, indicating that the sequenced P. chrysosporium mnp genes can be divided into three subfamilies on the basis of intron-exon structure. The mnp3 gene promoter contains putative metal response elements and heat shock elements which may be involved in the regulation of mnp gene transcription by Mn, the substrate for the enzyme, and by heat shock.

Purification and characterization of two manganese peroxidase isozymes from the white-rot basidiomycete Dichomitus squalens

Two manganese peroxidase isozymes, MnP1 and MnP2, were purified from the extracellular medium of ligninolytic cultures of Dichomitus squalens. The proteins were purified to homogeneity using DEAE-Sepharose chromatography and Mono Q fast protein liquid chromatography. MnP1 and MnP2 have molecular masses of 48000 and 48900 Da, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both isozymes are glycoproteins and each contains one iron protoporphyrin IX as a prosthetic group. The pl values of MnP1 and MnP2 are 4.15 and 3.90, respectively. N-Terminal amino-acid analysis suggests that these proteins are encoded by distinct genes. The Soret bands of the native ferric enzymes (408 nm and 406 nm, respectively) are shifted to 434 nm in the reduced enzymes and to 422 nm in the reduced-CO complexes. EPR g-values of the native enzymes are essentially identical to those for other MnPs and lignin peroxidases, and they confirm the high-spin state of the iron. The addition of 1 equivalent of H2O2 to either of the native ferric isozymes yields spectra which are characteristic of compound 1. Successive additions of 1 equivalent of ferrocyanide and 1 equivalent of H2O2 to the native enzymes yield spectra which are characteristic of compound II. Both MnP isozymes oxidize Mn2+ to Mn3+ in the presence of organic acid chelators. The MnP isozymes are produced by D. squalens only when the cells are grown in the presence of Mn.

Transformation of Phanerochaete chrysosporium and Neurospora crassa with adenine biosynthetic genes from Schizophyllum commune

SummaryProtoplasted basidiospores of two different adenine auxotrophs of the lignin-degrading basidiomycete Phanerochaete chrysosporium were transformed to prototrophy using plasmids containing genes encoding adenine biosynthetic enzymes from Schizophyllum commune. Fragments containing these genes were subcloned into pUC18 and P. chrysosporium transformants obtained with these subclones were analyzed. The subclones were mapped for restriction sites and the approximate locations of the complementing genes were determined. One of these plasmids was used to transform the Neurospora crassa auxotrophic strain ade2, thereby identifying the S. commune ade5 biosynthetic gene as encoding phosphoribosylaminoimidazole synthetase.

Lignin peroxidase from the basidiomycete Phanerochaete chrysosporium is synthesized as a preproenzyme

The cDNA clone L18 encoding lignin peroxidase LiP2, the most highly expressed LiP isozyme from Phanerochaete chrysosporium strain OGC101, was isolated and sequenced. Comparison of the cDNA sequence with the N-terminal sequence of the mature LiP2 protein isolated from culture medium suggests that the mature protein contains 343 amino acids (aa) and is preceded by a 28-aa leader sequence. In vitro transcription followed by in vitro translation and processing by signal peptidase resulted in cleavage at a site following the Ala21 (counted from the N-terminal Met1 of the initial translation product). The resultant protein contains a 7-aa propeptide, indicating that LiP is synthesized as a preproenzyme.

Ultrahigh (0.93A) resolution structure of manganese peroxidase from Phanerochaete chrysosporium: implications for the catalytic mechanism.

Manganese peroxidase (MnP) is an extracellular heme enzyme produced by the lignin-degrading white-rot fungus Phanerochaete chrysosporium. MnP catalyzes the peroxide-dependent oxidation of Mn(II) to Mn(III). The Mn(III) is released from the enzyme in complex with oxalate, enabling the oxalate-Mn(III) complex to serve as a diffusible redox mediator capable of oxidizing lignin, especially under the mediation of unsaturated fatty acids. One heme propionate and the side chains of Glu35, Glu39 and Asp179 have been identified as Mn(II) ligands in our previous crystal structures of native MnP. In our current work, new 0.93A and 1.05A crystal structures of MnP with and without bound Mn(II), respectively, have been solved. This represents only the sixth structure of a protein of this size at 0.93A resolution. In addition, this is the first structure of a heme peroxidase from a eukaryotic organism at sub-Angstrom resolution. These new structures reveal an ordering/disordering of the C-terminal loop, which is likely required for Mn binding and release. In addition, the catalytic Arg42 residue at the active site, normally thought to function only in the peroxide activation process, also undergoes ordering/disordering that is coupled to a transient H-bond with the Mn ligand, Glu39. Finally, these high-resolution structures also reveal the exact H atoms in several parts of the structure that are relevant to the catalytic mechanism.