The biosynthesis of benzoic acid glucosinolate esters in Arabidopsis thaliana.

The siliques and seeds of Arabidopsis thaliana accumulate a series of glucosinolates containing an alkyl side chain of varying length with a terminal benzoate ester function. The biosynthesis of these unusual nitrogen- and sulfur-containing natural products was investigated by feeding isotopically-labeled precursors to detached flowering stems. Glucosinolates were extracted, purified and analyzed by tandem mass spectrometry. Phenylalanine and benzoic acid were incorporated into the benzoate ester function, and methionine and acetate were incorporated into the aliphatic portion of the side chain in a position-specific manner. The labeling patterns observed were consistent with the chain extension of methionine by a three-step elongation cycle which begins with the condensation of acetyl-CoA with a 2-oxo acid derived from methionine and ends with an oxidative decarboxylation forming a new 2-oxo acid with an additional methylene group. Incorporation of desulfo-4-methylthiobutyl glucosinolate into 4-benzoyloxybutyl olucosinolate suggested chain-extended methionine derivatives are first converted to their corresponding methylthioalkyl glucosinolates with further side chain modification occurring later. Transformation of the methylthiol function to a hydroxyl group is followed by esterification with benzoic acid. The siliques appear to possess the complete machinery for carrying out all of the reactions in the biosyntheis of these complex glucosinolates.

Cloning and characterization of a second laccase gene from the lignin-degrading basidiomycete Pycnoporus cinnabarinus.

The gene lcc3-2 encoding a second laccase of the white-rot fungus Pycnoporus cinnabarinus has been cloned, sequenced, and characterized. The isolated gene consists of 2840bp, with the coding region interrupted by ten introns and flanked by an upstream region in which putative CAAT and TATA boxes were identified. The cDNA of lcc3-2 contains an open reading frame of 1563bp. The deduced mature laccase protein consisted of 498 amino acids and was preceded by a signal peptide of 23 amino acids. The sequence of lcc3-2 reveals 73% similarity on the protein level to the previously characterized lcc3-1. The new laccase gene shares highest similarity to lcc1 from Trametes villosa (75%), and lcc2 from the unidentified basidiomycete CECT 20197 (75%). The calculated isoelectric point (pI) of 6.1 for the gene product LCC3-2 was in good agreement with the experimentally determined pI of a laccase secreted by P. cinnabarinus grown on cellulose. Transcription analysis using competitive reverse transcription (RT)-PCR showed that lcc3-2 was expressed in glucose and cellulose containing cultures. However, in contrast to lcc3-1, lcc3-2 transcription was not increased in response to 2,5-xylidine.

Novel interaction between laccase and cellobiose dehydrogenase during pigment synthesis in the white rot fungus Pycnoporus cinnabarinus.

When glucose is the carbon source, the white rot fungus Pycnoporus cinnabarinus produces a characteristic red pigment, cinnabarinic acid, which is formed by laccase-catalyzed oxidation of the precursor 3-hydroxyanthranilic acid. When P. cinnabarinus was grown on media containing cellobiose or cellulose as the carbon source, the amount of cinnabarinic acid that accumulated was reduced or, in the case of cellulose, no cinnabarinic acid accumulated. Cellobiose-dependent quinone reducing enzymes, the cellobiose dehydrogenases (CDHs), inhibited the redox interaction between laccase and 3-hydroxyanthranilic acid. Two distinct proteins were purified from cellulose-grown cultures of P. cinnabarinus; these proteins were designated CDH I and CDH II. CDH I and CDH II were both monomeric proteins and had apparent molecular weights of about 81,000 and 101,000, respectively, as determined by both gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI values were approximately 5.9 for CDH I and 3.8 for CDH II. Both CDHs used several known CDH substrates as electron acceptors and specifically adsorbed to cellulose. Only CDH II could reduce cytochrome c. The optimum pH values for CDH I and CDH II were 5.5 and 4.5, respectively. In in vitro experiments, both enzymes inhibited laccase-mediated formation of cinnabarinic acid. Oxidation intermediates of 3-hydroxyanthranilic acid served as endogenous electron acceptors for the two CDHs from P. cinnabarinus. These results demonstrated that in the presence of a suitable cellulose-derived electron donor, CDHs can regenerate fungal metabolites oxidized by laccase, and they also supported the hypothesis that CDHs act as links between cellulolytic and ligninolytic pathways.

Molecular analysis of a laccase gene from the white rot fungus Pycnoporus cinnabarinus.

It was recently shown that the white rot basidiomycete Pycnoporus cinnabarinus secretes an unusual set of phenoloxidases when it is grown under conditions that stimulate ligninolysis (C. Eggert, U. Temp, and K.-E. L. Eriksson, Appl. Environ. Microbiol. 62:1151-1158, 1996). In this report we describe the results of a cloning and structural analysis of the laccase-encoding gene (lcc3-1) expressed by P. cinnabarinus during growth under xylidine-induced conditions. The coding region of the genomic laccase sequence, which is preceded by the eukaryotic promoter elements TATA and CAATA, spans more than 2,390 bp. The corresponding laccase cDNA was identical to the genomic sequence except for 10 introns that were 50 to 60 bp long. A sequence analysis indicated that the P. cinnabarinus lcc3-1 product has a Phe residue at a position likely to influence the reduction-oxidation potential of the enzyme's type 1 copper center. The P. cinnabarinus lcc3-1 sequence was most similar to the sequence encoding a laccase from Coriolus hirsutus (level of similarity, 84%).

A small-scale method for screening of lignin-degrading microorganisms.

A new method to facilitate rapid screening of lignin-degrading microorganisms was developed. Fungal strains are cultivated in tissue culture plates containing C-ring-labeled dehydrogenation polymerizate (DHP) (synthetic lignin). Evolved CO(2) is trapped in barium-saturated filter paper and is detected by exposing the paper to X-ray film. Analysis of the autoradiograms, carried out by density measurement with an image analysis program, allows for a semiquantitative estimation of the amount of CO(2) evolved. The method is especially useful for screening for new, powerful lignin-degrading strains in both man-made and natural environments. It eliminates the need for special equipment for their cultivation and trapping of CO(2) as well as laborious sample analysis. The method has in this study been used to test three novel fungal isolates and a laccaseless mutant of the basidiomycete Pycnoporus cinnabarinus. Their ligninolytic capacities were compared with those of the potent lignin degrader Ceriporiopsis subvermispora.

Laccase is essential for lignin degradation by the white-rot fungus Pycnoporus cinnabarinus.

The white-rot fungus, Pycnoporus cinnabarinus, provides an excellent model organism to elucidate the controversial role of laccase in lignin degradation. P. cinnabarinus produces laccase in one isoform as the predominant phenoloxidase in ligninolytic cultures, and neither LiP nor MnP are secreted. Yet, P. cinnabarinus degrades lignin very efficiently. In the present work, we show that laccase-less mutants of P. cinnabarinus were greatly reduced in their ability to metabolize 14C ring-labeled DHP. However, 14CO2 evolution in these mutant cultures could be restored to levels comparable to those of the wild-type cultures by addition of purified P. cinnabarinus laccase. This clearly indicates that laccase is absolutely essential for lignin degradation by P. cinnabarinus.

A fungal metabolite mediates degradation of non-phenolic lignin structures and synthetic lignin by laccase.

Lignin peroxidase is generally considered to be a primary catalyst for oxidative depolymerization of lignin by white-rot fungi. However, some white-rot fungi lack lignin peroxidase. Instead, many produce laccase, even though the redox potentials of known laccases are too low to directly oxidize the non-phenolic components of lignin. Pycnoporus cinnabarinus is one example of a laccase-producing fungus that degrades lignin very efficiently. To overcome the redox potential barrier, P. cinnabarinus produces a metabolite, 3-hydroxyanthranilate that can mediate the oxidation of how non-phenolic substrates by laccase. This is the first description of how laccase might function in a biological system for the complete depolymerization of lignin.

The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus: purification and characterization of the laccase.

The white rot fungus Pycnoporus cinnabarinus was characterized with respect to its set of extracellular phenoloxidases. Laccase was produced as the predominant extracellular phenoloxidase in conjunction with low amounts of an unusual peroxidase. Neither lignin peroxidase nor manganese peroxidase was detected. Laccase was produced constitutively during primary metabolism. Addition of the most effective inducer, 2,5-xylidine, enhanced laccase production ninefold without altering the isoenzyme pattern of the enzyme. Laccase purified to apparent homogeneity was a single polypeptide having a molecular mass of approximately 81,000 Da, as determined by calibrated gel filtration chromatography, and a carbohydrate content of 9%. The enzyme displayed an unusual behavior on isoelectric focusing gels; the activity was split into one major band (pI, 3.7) and several minor bands of decreasing intensity which appeared at regular, closely spaced intervals toward the alkaline end of the gel. Repeated electrophoresis of the major band under identical conditions produced the same pattern, suggesting that the laccase was secreted as a single acidic isoform with a pI of about 3.7 and that the multiband pattern was an artifact produced by electrophoresis. This appeared to be confirmed by N-terminal amino acid sequencing of the purified enzyme, which yielded a single sequence for the first 21 residues. Spectroscopic analysis indicated a typical laccase active site in the P. cinnabarinus enzyme since all three typical Cu(II)-type centers were identified. Substrate specificity and inhibitor studies also indicated the enzyme to be a typical fungal laccase. The N-terminal amino acid sequence of the P. cinnabarinus laccase showed close homology to the N-terminal sequences determined for laccases from Trametes versicolor, Coriolus hirsutus, and an unidentified basidiomycete, PM1. The principal features of the P. cinnabarinus enzyme system, a single predominant laccase and a lack of lignin- or manganese-type peroxidase, make this organism an interesting model for further studies of possible alternative pathways of lignin degradation by white rot fungi.

Laccase-mediated formation of the phenoxazinone derivative, cinnabarinic acid.

The phenoxazinone chromophore occurs in a variety of biological systems, including numerous pigments and certain antibiotics. It also appears to form as part of a mechanism to protect mammalian tissue from oxidative damage. During cultivation of the basidiomycete, Pycnoporus cinnabarinus, a red pigment was observed to accumulate in the culture medium. It was identified as the phenoxazinone derivative, cinnabarinic acid (CA). Laccase was the predominant extracellular phenoloxidase activity in P. cinnabarinus cultures. In vitro studies showed that CA was formed after oxidation of the precursor, 3-hydroxyanthranilic acid (3-HAA), by laccases. Moreover, oxidative coupling of 3-HAA to form CA was also demonstrated for the mammalian counterpart of laccase, the blue copper oxidase, ceruloplasmin.