Structures of Phanerochaete chrysosporium Cel7D in complex with product and inhibitors.
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The cellobiohydrolase Pc_Cel7D is the major cellulase produced by the white-rot fungus Phanerochaete chrysosporium, constituting approximately 10% of the total secreted protein in liquid culture on cellulose. The enzyme is classified into family 7 of the glycoside hydrolases and, like other family members, catalyses cellulose hydrolysis with net retention of the anomeric carbon configuration. Previous work described the apo structure of the enzyme. Here we investigate the binding of the product, cellobiose, and several inhibitors, i.e. lactose, cellobioimidazole, Tris/HCl, calcium and a thio-linked substrate analogue, methyl 4-S-beta-cellobiosyl-4-thio-beta-cellobioside (GG-S-GG). The three disaccharides bind in the glucosyl-binding subsites +1 and +2, close to the exit of the cellulose-binding tunnel/cleft. Pc_Cel7D binds to lactose more strongly than cellobiose, while the opposite is true for the homologous Trichoderma reesei cellobiohydrolase Tr_Cel7A. Although both sugars bind Pc_Cel7D in a similar fashion, the different preferences can be explained by varying interactions with nearby loops. Cellobioimidazole is bound at a slightly different position, displaced approximately 2 A toward the catalytic centre. Thus the Pc_Cel7D complexes provide evidence for two binding modes of the reducing-end cellobiosyl moiety; this conclusion is confirmed by comparison with other available structures. The combined results suggest that hydrolysis of the glycosyl-enzyme intermediate may not require the prior release of the cellobiose product from the enzyme. Further, the structure obtained in the presence of both GG-S-GG and cellobiose revealed electron density for Tris at the catalytic centre. Inhibition experiments confirm that both Tris and calcium are effective inhibitors at the conditions used for crystallization. (+info)
An exo-beta-1,3-galactanase having a novel beta-1,3-galactan-binding module from Phanerochaete chrysosporium.
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An exo-beta-1,3-galactanase gene from Phanerochaete chrysosporium has been cloned, sequenced, and expressed in Pichia pastoris. The complete amino acid sequence of the exo-beta-1,3-galactanase indicated that the enzyme consists of an N-terminal catalytic module with similarity to glycoside hydrolase family 43 and an additional unknown functional domain similar to carbohydrate-binding module family 6 (CBM6) in the C-terminal region. The molecular mass of the recombinant enzyme was estimated as 55 kDa based on SDS-PAGE. The enzyme showed reactivity only toward beta-1,3-linked galactosyl oligosaccharides and polysaccharide as substrates but did not hydrolyze beta-1,4-linked galacto-oligosaccharides, beta-1,6-linked galacto-oligosaccharides, pectic galactan, larch arabinogalactan, arabinan, gum arabic, debranched arabinan, laminarin, soluble birchwood xylan, or soluble oat spelled xylan. The enzyme also did not hydrolyze beta-1,3-galactosyl galactosaminide, beta-1,3-galactosyl glucosaminide, or beta-1,3-galactosyl arabinofuranoside, suggesting that it specifically cleaves the internal beta-1,3-linkage of two galactosyl residues. High performance liquid chromatographic analysis of the hydrolysis products showed that the enzyme produced galactose from beta-1,3-galactan in an exo-acting manner. However, no activity toward p-nitrophenyl beta-galactopyranoside was detected. When incubated with arabinogalactan proteins, the enzyme produced oligosaccharides together with galactose, suggesting that it is able to bypass beta-1,6-linked galactosyl side chains. The C-terminal CBM6 did not show any affinity for known substrates of CBM6 such as xylan, cellulose, and beta-1,3-glucan, although it bound beta-1,3-galactan when analyzed by affinity electrophoresis. Frontal affinity chromatography for the CBM6 moiety using several kinds of terminal galactose-containing oligosaccharides as the analytes clearly indicated that the CBM6 specifically interacted with oligosaccharides containing a beta-1,3-galactobiose moiety. When the degree of polymerization of galactose oligomers was increased, the binding affinity of the CBM6 showed no marked change. (+info)
Electron transfer chain reaction of the extracellular flavocytochrome cellobiose dehydrogenase from the basidiomycete Phanerochaete chrysosporium.
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Cellobiose dehydrogenase (CDH) is an extracellular flavocytochrome containing flavin and b-type heme, and plays a key role in cellulose degradation by filamentous fungi. To investigate intermolecular electron transfer from CDH to cytochrome c, Phe166, which is located in the cytochrome domain and approaches one of propionates of heme, was mutated to Tyr, and the thermodynamic and kinetic properties of the mutant (F166Y) were compared with those of the wild-type (WT) enzyme. The mid-point potential of heme in F166Y was measured by cyclic voltammetry, and was estimated to be 25 mV lower than that of WT at pH 4.0. Although presteady-state reduction of flavin was not affected by the mutation, the rate of subsequent electron transfer from flavin to heme was halved in F166Y. When WT or F166Y was reduced with cellobiose and then mixed with cytochrome c, heme re-oxidation and cytochrome c reduction occurred synchronously, suggesting that the initial electron is transferred from reduced heme to cytochrome c. Moreover, in both enzymes the observed rate of the initial phase of cytochrome c reduction was concentration dependent, whereas the second phase of cytochrome c reduction was dependent on the rate of electron transfer from flavin to heme, but not on the cytochrome c concentration. In addition, the electron transfer rate from flavin to heme was identical to the steady-state reduction rate of cytochrome c in both WT and F166Y. These results clearly indicate that the first and second electrons of two-electron-reduced CDH are both transferred via heme, and that the redox reaction of CDH involves an electron-transfer chain mechanism in cytochrome c reduction. (+info)
Genome-wide structural and evolutionary analysis of the P450 monooxygenase genes (P450ome) in the white rot fungus Phanerochaete chrysosporium: evidence for gene duplications and extensive gene clustering.
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BACKGROUND: Phanerochaete chrysosporium, the model white rot basidiomycetous fungus, has the extraordinary ability to mineralize (to CO2) lignin and detoxify a variety of chemical pollutants. Its cytochrome P450 monooxygenases have recently been implied in several of these biotransformations. Our initial P450 cloning efforts in P. chrysosporium and its subsequent whole genome sequencing have revealed an extraordinary P450 repertoire ("P450ome") containing at least 150 P450 genes with yet unknown function. In order to understand the functional diversity and the evolutionary mechanisms and significance of these hemeproteins, here we report a genome-wide structural and evolutionary analysis of the P450ome of this fungus. RESULTS: Our analysis showed that P. chrysosporium P450ome could be classified into 12 families and 23 sub-families and is characterized by the presence of multigene families. A genome-level structural analysis revealed 16 organizationally homogeneous and heterogeneous clusters of tandem P450 genes. Analysis of our cloned cDNAs revealed structurally conserved characteristics (intron numbers and locations, and functional domains) among members of the two representative multigene P450 families CYP63 and CYP505 (P450foxy). Considering the unusually complex structural features of the P450 genes in this genome, including microexons (2-10 aa) and frequent small introns (45-55 bp), alternative splicing, as experimentally observed for CYP63, may be a more widespread event in the P450ome of this fungus. Clan-level phylogenetic comparison revealed that P. chrysosporium P450 families fall under 11 fungal clans and the majority of these multigene families appear to have evolved locally in this genome from their respective progenitor genes, as a result of extensive gene duplications and rearrangements. CONCLUSION: P. chrysosporium P450ome, the largest known to date among fungi, is characterized by tandem gene clusters and multigene families. This enormous P450 gene diversity has evolved by extensive gene duplications and intragenomic recombinations of the progenitor genes presumably to meet the exceptionally high metabolic demand of this biodegradative group of basidiomycetous fungi in ecological niches. In this context, alternative splicing appears to further contribute to the evolution of functional diversity of the P450ome in this fungus. The evolved P450 diversity is consistent with the known vast biotransformation potential of P. chrysosporium. The presented analysis will help design future P450 functional studies to understand the underlying mechanisms of secondary metabolism and oxidative biotransformation pathways in this model white rot fungus. (+info)
Meiotic segregation analysis of restriction site polymorphisms allows rapid genetic mapping.
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The white rot fungus Phanerochaete chrysosporium is an efficient degrader of lignin. Development of classical genetics in this organism would require several years of work, so an alternative strategy was devised. As in the mapping of human genetic diseases, the approach depends on the presence of interallelic restriction site polymorphism, but differs in following their meiotic segregation into the haploid state. Using a set of 20 cloned sequences as probes we were able to demonstrate meiotic segregation of 14 restriction site polymorphisms from fruiting of a single dikaryotic culture and obtained evidence of genetic linkage within two groups of three and two of two. The method could be used to obtain a genetic map of this fungus and should be applicable to genetic analysis of any other organism for which haploid material is available. (+info)
Characterization of carbohydrate-binding cytochrome b562 from the white-rot fungus Phanerochaete chrysosporium.
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cDNA encoding a hemoprotein similar to the cytochrome domain of extracellular flavocytochrome cellobiose dehydrogenase (CDH) was cloned from the white-rot fungus Phanerochaete chrysosporium. The deduced amino acid sequence implies that there is a two-domain structure consisting of an N-terminal cytochrome domain and a C-terminal family 1 carbohydrate-binding module (CBM1) but that the flavin-containing domain of CDH is not present. The gene transcripts were observed in cultures in cellulose medium but not in cultures in glucose medium, suggesting that there is regulation by carbon catabolite repression. The gene was successfully overexpressed in Pichia pastoris, and the recombinant protein was designated carbohydrate-binding cytochrome b562 (CBCyt. b562). The resonance Raman spectrum suggested that the heme of CBCyt. b562 is 6-coordinated in both the ferric and ferrous states. Moreover, the redox potential measured by cyclic voltammetry was similar to that of the cytochrome domain of CDH. These results suggest that the redox characteristics may be similar to those of the cytochrome domain of CDH, and so CBCyt. b562 may have an electron transfer function. In a binding study with various carbohydrates, CBCyt. b562 was adsorbed with high affinity on both cellulose and chitin. As far as we know, this is the first example of a CBM1 connected to a domain without apparent catalytic activity for carbohydrate; this CBM1 may play a role in localization of the redox protein on the surface of cellulose or on the fungal sheath in vivo. (+info)
Molecular cloning and characterization of a peroxiredoxin from Phanerochaete chrysosporium.
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Peroxiredoxins (Prxs) are a ubiquitous family of peroxidases widely distributed among prokaryotes and eukaryotes. Here, we report on the cloning and functional characterization of a cDNA designated PcPrx-1, encoding peroxiredoxin from the white-rot fungus Phanerochaete chrysosporium. The full-length PcPrx-1 cDNA (932 bp) contains an open reading frame of 200 amino acid residues with a molecular mass of 22.1 kDa. The deduced primary structure of PcPrx-1 polypeptide shows a high level of sequence identity to other recently identified 2-cys peroxiredoxins. The recombinant PcPrx-1 protein was expressed as a histidine fusion protein in Escherichia coli and purified with a Ni2+-column. The purified protein was shown to have a protective effect against plasmid DNA cleavage by reactive oxygen species. The PcPrx-1 protein displays the ability to remove H2O2 in a ferrithiocyanate system. The results of this study suggest that PcPrx-1 may play a protective role against oxidative stress in P. chrysosporium. (+info)
Estrogenic reduction of styrene monomer degraded by Phanerochaete chrysosporium KFRI 20742.
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The characteristic biodegradation of monomeric styrene by Phanerochaete chrysosporium KFRI 20742, Trametes versicolor KFRI 20251 and Daldinia concentrica KFRI 40-1 was carried out to examine the resistance, its degradation efficiency and metabolites analysis. The estrogenic reduction effect of styrene by the fungi was also evaluated. The mycelium growth of fungi differentiated depending on the concentration levels of styrene. Additionally P. chrysosporium KFRI 20742 showed superior mycelium growth at less than 200 mg/l, while D. concentrica KFRI 40-1 was more than 200 mg/l. The degradation efficiency reached 99% during one day of incubation for all the fungi. Both manganese-dependent peroxidase and laccase activities in liquid medium were the highest at the initial stage of incubation, whereas the lowest was after the addition of styrene. However, both activities were gradually recovered after. The major metabolites of styrene by P. chrysosporium KFRI 20742 were 2-phenyl ethanol, benzoic acid, cyclohexadiene-1,4-dione, butanol and succinic acid. From one to seven days of incubating the fungi, the expression of pS2 mRNA widely known as an estrogen response gene was decreased down to the level of baseline after one day. Also, the estrogenic effect of styrene completely disappeared after treatment with supernatant of P. chrysosporium KFRI 20742 from one week of culture down to the levels of vehicle. (+info)