The nuclear ribosomal DNA intergenic spacer as a target sequence to study intraspecific diversity of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum directly on pinus root systems.
Polymorphism of the nuclear ribosomal DNA intergenic spacer (IGS) of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum was studied to evaluate whether this sequence could be used in field studies to estimate the diversity of strains forming mycorrhizas on individual Pinus pinaster root systems. This sequence was amplified by PCR from 125 haploid homokaryotic strains collected in 14 P. pinaster stands along the Atlantic coast of France by using conserved oligonucleotide primers. Restriction enzyme digestion of the amplified 3.4-kbp-long IGS allowed us to characterize 24 alleles whose frequencies differed. Nine of these alleles were found only once, whereas about 60% of the strains contained four of the alleles. Local populations could be almost as diverse as the entire population along a 150-km stretch of coastline that was examined; for example, 13 alleles were found in a single forest stand. The IGS from one strain was partially sequenced, and the sequence data were used to design oligonucleotides which allowed separate PCR amplification of three different segments of the IGS. Most polymorphisms observed among the full-length IGS regions resulted from polymorphisms in an internal ca. 1,500-bp-long sequence characterized by length variations that may have resulted from variable numbers of a T2AG3 motif. This internal polymorphic sequence could not be amplified from the genomes of nine other Hebeloma species. Analysis of this internal sequence amplified from the haploid progenies of 10 fruiting bodies collected in a 70-m2 area resulted in identification of six allelic forms and seven distinct diplotypes out of the 21 possible different combinations. Moreover, optimization of the PCR conditions resulted in amplification of this sequence from more than 80% of the DNA samples extracted from individual H. cylindrosporum infected P. pinaster mycorrhizal root tips, thus demonstrating the usefulness of this sequence for studying the below-ground diversity of mycorrhizas formed by genets belonging to the same fungal species. (+info)
Cloning and characterization of a cDNA encoding a novel extracellular peroxidase from Trametes versicolor.
The white rot basidiomycete Trametes versicolor secretes a large number of peroxidases which are believed to be involved in the degradation of polymeric lignin. These peroxidases have been classified previously as lignin peroxidases or manganese peroxidases (MnP). We have isolated a novel extracellular peroxidase-encoding cDNA sequence from T. versicolor CU1, the transcript levels of which are repressed by low concentrations of Mn2+ and induced by nitrogen and carbon but not induced in response to a range of stresses which have been reported to induce MnP expression. (+info)
Direct interaction of lignin and lignin peroxidase from Phanerochaete chrysosporium.
Binding properties of lignin peroxidase (LiP) from the basidiomycete Phanerochaete chrysosporium against a synthetic lignin (dehydrogenated polymerizate, DHP) were studied with a resonant mirror biosensor. Among several ligninolytic enzymes, only LiP specifically binds to DHP. Kinetic analysis revealed that the binding was reversible, and that the dissociation equilibrium constant was 330 microM. The LiP-DHP interaction was controlled by the ionization group with a pKa of 5.3, strongly suggesting that a specific amino acid residue plays a role in lignin binding. A one-electron transfer from DHP to oxidized intermediates LiP compounds I and II (LiPI and LiPII) was characterized by using a stopped-flow technique, showing that binding interactions of DHP with LiPI and LiPII led to saturation kinetics. The dissociation equilibrium constants for LiPI-DHP and LiPII-DHP interactions were calculated to be 350 and 250 microM, and the first-order rate constants for electron transfer from DHP to LiPI and to LiPII were calculated to be 46 and 16 s-1, respectively. These kinetic and spectral studies strongly suggest that LiP is capable of oxidizing lignin directly at the protein surface by a long-range electron transfer process. A close look at the crystal structure suggested that LiP possesses His-239 as a possible lignin-binding site on the surface, which is linked to Asp-238. This Asp residue is hydrogen-bonded to the proximal His-176. This His-Asp...proximal-His motif would be a possible electron transfer route to oxidize polymeric lignin. (+info)
A different approach to treatment of phenylketonuria: phenylalanine degradation with recombinant phenylalanine ammonia lyase.
Phenylketonuria (PKU), with its associated hyperphenylalaninemia (HPA) and mental retardation, is a classic genetic disease and the first to have an identified chemical cause of impaired cognitive development. Treatment from birth with a low phenylalanine diet largely prevents the deviant cognitive phenotype by ameliorating HPA and is recognized as one of the first effective treatments of a genetic disease. However, compliance with dietary treatment is difficult and when it is for life, as now recommended by an internationally used set of guidelines, is probably unrealistic. Herein we describe experiments on a mouse model using another modality for treatment of PKU compatible with better compliance using ancillary phenylalanine ammonia lyase (PAL, EC 126.96.36.199) to degrade phenylalanine, the harmful nutrient in PKU; in this treatment, PAL acts as a substitute for the enzyme phenylalanine monooxygenase (EC 188.8.131.52), which is deficient in PKU. PAL, a robust enzyme without need for a cofactor, converts phenylalanine to trans-cinnamic acid, a harmless metabolite. We describe (i) an efficient recombinant approach to produce PAL enzyme, (ii) testing of PAL in orthologous N-ethyl-N'-nitrosourea (ENU) mutant mouse strains with HPA, and (iii) proofs of principle (PAL reduces HPA)-both pharmacologic (with a clear dose-response effect vs. HPA after PAL injection) and physiologic (protected enteral PAL is significantly effective vs. HPA). These findings open another way to facilitate treatment of this classic genetic disease. (+info)
Molecular gene organisation and secondary structure of the mitochondrial large subunit ribosomal RNA from the cultivated Basidiomycota Agrocybe aegerita: a 13 kb gene possessing six unusual nucleotide extensions and eight introns.
The complete gene sequence and secondary structure of the mitochondrial LSU rRNA from the cultivated Basidiomycota Agrocybe aegerita was derived by chromosome walking. The A.aegerita LSU rRNA gene (13 526 nt) represents, to date, the longest described, due to the highest number of introns (eight) and the occurrence of six long nucleotidic extensions. Seven introns belong to group I, while the intronic sequence i5 constitutes the first typical group II intron reported in a fungal mitochondrial LSU rDNA. As with most fungal LSU rDNA introns reported to date, four introns (i5-i8) are distributed in domain V associated with the peptidyl-transferase activity. One intron (i1) is located in domain I, and three (i2-i4) in domain II. The introns i2-i8 possess homologies with other fungal, algal or protozoan introns located at the same position in LSU rDNAs. One of them (i6) is located at the same insertion site as most Ascomycota or algae LSU introns, suggesting a possible inheritance from a common ancestor. On the contrary, intron i1 is located at a so-far unreported insertion site. Among the six unusual nucleotide extensions, five are located in domain I and one in domain V. This is the first report of a mitochondrial LSU rRNA gene sequence and secondary structure for the whole Basidiomycota division. (+info)
Lignocellulose degradation by Phanerochaete chrysosporium: purification and characterization of the main alpha-galactosidase.
The main alpha-galactosidase was purified to homogeneity, in 30% yield, from a solid culture of Phanerochaete chrysosporium on 1 part wheat bran/2 parts thermomechanical softwood pulp. It is a glycosylated tetramer of 50 kDa peptide chains, which gives the N-terminal sequence ADNGLAITPQMG(?W)NT(?W)NHFG(?W)DIS(?W)DTI. It is remarkably stable, with crude extracts losing no activity over 3 h at 80 degrees C, and the purified enzyme retaining its activity over several months at 4 degrees C. The kinetics of hydrolysis at 25 degrees C of various substrates by this retaining enzyme were measured, absolute parameters being obtained by active-site titration with 2',4',6'-trinitrophenyl 2-deoxy-2, 2-difluoro-alpha-D-galactopyranoside. The variation of kcat/Km for 1-naphthyl-alpha-D-galactopyranoside with pH is bell-shaped, with pK1=1.91 and pK2=5.54. The alphaD(V/K) value for p-nitrophenyl-alpha-D-glucopyranoside is 1.031+/-0.007 at the optimal pH of 3.75 and 1.114+/-0.006 at pH7.00, indicating masking of the intrinsic effect at optimal pH. There is no alpha-2H effect on binding galactose [alphaD(Ki)=0.994+/-0.013]. The enzyme hydrolyses p-nitrophenyl beta-L-arabinopyranoside approximately 510 times slower than the galactoside, but has no detectable activity on the alpha-D-glucopyranoside or alpha-D-mannopyranoside. Hydrolysis of alpha-galactosides with poor leaving groups is Michaelian, but that of substrates with good leaving groups exhibits pronounced apparent substrate inhibition, with Kis values similar to Km values. We attribute this to the binding of the second substrate molecule to a beta-galactopyranosyl-enzyme intermediate, forming an E.betaGal. alphaGalX complex which turns over slowly, if at all. 1-Fluoro-alpha-D-galactopyranosyl fluoride, unlike alpha-D-galactopyranosyl fluoride, is a Michaelian substrate, indicating that the effect of 1-fluorine substitution is greater on the first than on the second step of the enzyme reaction. (+info)
Biodegradative mechanism of the brown rot basidiomycete Gloeophyllum trabeum: evidence for an extracellular hydroquinone-driven fenton reaction.
We have identified key components of the extracellular oxidative system that the brown rot fungus Gloeophyllum trabeum uses to degrade a recalcitrant polymer, polyethylene glycol, via hydrogen abstraction reactions. G. trabeum produced an extracellular metabolite, 2,5-dimethoxy-1,4-benzoquinone, and reduced it to 2,5-dimethoxyhydroquinone. In the presence of 2,5-dimethoxy-1,4-benzoquinone, the fungus also reduced extracellular Fe3+ to Fe2+ and produced extracellular H2O2. Fe3+ reduction and H2O2 formation both resulted from a direct, non-enzymatic reaction between 2,5-dimethoxyhydroquinone and Fe3+. Polyethylene glycol depolymerization by G. trabeum required both 2,5-dimethoxy-1,4-benzoquinone and Fe3+ and was completely inhibited by catalase. These results provide evidence that G. trabeum uses a hydroquinone-driven Fenton reaction to cleave polyethylene glycol. We propose that similar reactions account for the ability of G. trabeum to attack lignocellulose. (+info)
Aromatic ring cleavage of a non-phenolic beta-O-4 lignin model dimer by laccase of Trametes versicolor in the presence of 1-hydroxybenzotriazole.
The novel cleavage products, 2,3-dihydroxy-1-(4-ethoxy-3-methoxyphenyl)-1-formyloxypropane (II) and 1-(4-ethoxy-3-methoxyphenyl)-1,2,3-trihydroxypropane-2,3-cyclic carbonate (III) were identified as products of a non-phenolic beta-O-4 lignin model dimer, 1,3-dihydroxy-2-(2,6-dimethoxylphenoxy)-1-(4-ethoxy-3-methoxypheny l)propane (I), by a Trametes versicolor laccase in the presence of 1-hydroxybenzotriazole (1-HBT). An isotopic experiment with a 13C-labeled lignin model dimer, 1,3-dihydroxy-2-(2,6-[U-ring-13C] dimethoxyphenoxy)-1-(4-ethoxy-3-methoxyphenyl)propane (I-13C) indicated that the formyl and carbonate carbons of products II and III were derived from the beta-phenoxy group of beta-O-4 lignin model dimer I as aromatic ring cleavage fragments. These results show that the laccase-1-HBT couple could catalyze the aromatic ring cleavage of non-phenolic beta-O-4 lignin model dimer in addition to the beta-ether cleavage, Calpha-Cbeta cleavage, and Calpha-oxidation. (+info)