A comparative structural analysis of the surface properties of asco-laccases.
ABSTRACT: Laccases of different biological origins have been widely investigated and these studies have elucidated fundamentals of the generic catalytic mechanism. However, other features such as surface properties and residues located away from the catalytic centres may also have impact on enzyme function. Here we present the crystal structure of laccase from Myceliophthora thermophila (MtL) to a resolution of 1.62 Å together with a thorough structural comparison with other members of the CAZy family AA1_3 that comprises fungal laccases from ascomycetes. The recombinant protein produced in A. oryzae has a molecular mass of 75 kDa, a pI of 4.2 and carries 13.5 kDa N-linked glycans. In the crystal, MtL forms a dimer with the phenolic substrate binding pocket blocked, suggesting that the active form of the enzyme is monomeric. Overall, the MtL structure conforms with the canonical fold of fungal laccases as well as the features specific for the asco-laccases. However, the structural comparisons also reveal significant variations within this taxonomic subgroup. Notable differences in the T1-Cu active site topology and polar motifs imply molecular evolution to serve different functional roles. Very few surface residues are conserved and it is noticeable that they encompass residues that interact with the N-glycans and/or are located at domain interfaces. The N-glycosylation sites are surprisingly conserved among asco-laccases and in most cases the glycan displays extensive interactions with the protein. In particular, the glycans at Asn88 and Asn210 appear to have evolved as an integral part of the asco-laccase structure. An uneven distribution of the carbohydrates around the enzyme give unique properties to a distinct part of the surface of the asco-laccases which may have implication for laccase function-in particular towards large substrates.
Project description:Typical laccases have four copper atoms, which form three different copper centers, of which the T1 copper is responsible for the blue color of the enzyme and gives it a characteristic absorbance around 610 nm. Several laccases have unusual spectral properties and are referred to as yellow or white laccases. Only two yellow laccases from the Ascomycota phylum have been described previously, and only one amino acid sequence of those enzymes is available. A yellow laccase Bcl1 from <i>Botrytis cinerea</i> strain 241 has been identified, purified and characterized in this work. The enzyme appears to be a dimer with a molecular mass of 186 kDa. The gene encoding the Bcl1 protein has been cloned, and the sequence analysis shows that the yellow laccase Bcl1 is phylogenetically distinct from other known yellow laccases. In addition, a comparison of amino acid sequences, and 3D modeling shows that the Bcl1 laccase lacks a conservative tyrosine, which is responsible for absorption quenching at 610 nm in another yellow asco-laccase from <i>Sclerotinia sclerotiorum</i>. High thermostability, high salt tolerance, broad substrate specificity, and the ability to decolorize dyes without the mediators suggest that the Bcl1 laccase is a potential enzyme for various industrial applications.
Project description:A high-efficiency laccase, DLac, was isolated from Cerrena sp. RSD1. The kinetic studies indicate that DLac is a diffusion-limited enzyme. The crystal structure of DLac was determined to atomic resolution, and its overall structure shares high homology to monomeric laccases, but displays unique substrate-binding loops from those in other laccases. The substrate-binding residues with small side chain and the short substrate-binding loop IV broaden the substrate-binding cavity and may facilitate large substrate diffusion. Unlike highly glycosylated fungal laccases, the less-glycosylated DLac contains one highly conserved glycosylation site at N432 and an unique glycosylation site at N468. The N-glycans stabilize the substrate-binding loops and the protein structure, and the first N-acetylglucosamine is crucial for the catalytic efficiency. Additionally, a fivefold increase in protein yield is achieved via the submerged culture method for industrial applications.The atomic coordinates of the structure of DLac from Cerrena sp. RSD1 and structural factors have been deposited in the RCSB Protein Data Bank (PDB ID: 5Z1X).
Project description:Agaricomycetes fungi responsible for decay of wood and other lignocellulosic substrates constitute a valuable source of lignin-degrading enzymes. Among these enzymes, laccases (multi-copper oxidases) present remarkable biotechnological potential as environmentally friendly biocatalysts able to oxidize a wide range of aromatic compounds using oxygen as the only requirement. Laccases from saprotrophic Agaricales species have been much less studied than laccases from Polyporales, despite the fact that the former fungi are excellent sources of laccases. Here, the gene of a novel laccase of <i>Agrocybe pediades</i>, that is secreted by the fungus during lignocellulose degradation, was synthesised de novo and expressed in <i>Saccharomyces cerevisiae</i> using an improved signal peptide previously obtained and enzyme directed evolution. The characterization of the new laccase variants provided new insights on the contribution of different amino acid residues to modulate laccase production, catalytic activity or optimal pH. The selected double-mutated variant also showed interesting properties as a biocatalyst, such as the ability to oxidise a wide range of substrates, including high-redox potential mediators and recalcitrant organic dyes, improved activity at neutral pH and high tolerance to inhibitors. Finally, we demonstrate the existence of three <i>N</i>-glycosylation sites in the laccase and their distinct effect on the secretion or catalytic activity of the enzyme.
Project description:Background:Phenolic compounds, which are produced routinely by industrial and urban activities, possess dangers to live organisms and environment. Laccases are oxidoreductase enzymes with the ability of remediating a wide variety of phenolic compounds to more benign molecules. The purpose of the present research is surface display of a laccase enzyme with adhesin involved in diffuse adhesion (AIDA-I) autotransporter system on the surface of Escherichia coli cells for bioremediation of phenolic compounds. Methods:The expression of laccase was regulated by a phenol-responsive promoter (a ?54 promoter). The constitutively-expressed CapR transcription activator was able to induce laccase expression in the presence of phenolic compounds. Results:Western blot analysis showed the expression and correct transfer of the enzyme to the outer membrane of E. coli cells in the presence of phenol. Activity assay confirmed the correct folding of the enzyme after translocation through the autotransporter system. HPLC analysis of residual phenol in culture medium showed a significant reduction of phenol concentration in the presence of cells displaying laccase on the surface. Conclusion:Our findings confirm that autodisplay enables functional surface display of laccase for direct substrate-enzyme availability by overcoming membrane hindrance.
Project description:Laccases are copper-containing enzymes which oxidize phenolic substrates and transfer the electrons to oxygen. Many filamentous fungi contain several laccase-encoding genes, but their biological roles are mostly not well understood. The main interest in laccases in biotechnology is their potential to be used to detoxify phenolic substances. We report here on a novel application of laccases as a reporter system in fungi. We purified a laccase enzyme from the ligno-cellulolytic ascomycete Stachybotrys chartarum. It oxidized the artificial substrate 2,2'-azino-di-(3-ethylbenzthiazolinsulfonate) (ABTS). The corresponding gene was isolated and expressed in Aspergillus nidulans, Aspergillus niger, and Trichoderma reesei. Heterologously expressed laccase activity was monitored in colorimetric enzyme assays and on agar plates with ABTS as a substrate. The use of laccase as a reporter was shown in a genetic screen for the isolation of improved T. reesei cellulase production strains. In addition to the laccase from S. charatarum, we tested the application of three laccases from A. nidulans (LccB, LccC, and LccD) as reporters. Whereas LccC oxidized ABTS (Km = 0.3 mM), LccD did not react with ABTS but with DMA/ADBP (3,5-dimethylaniline/4-amino-2,6-dibromophenol). LccB reacted with DMA/ADBP and showed weak activity with ABTS. The different catalytic properties of LccC and LccD allow simultaneous use of these two laccases as reporters in one fungal strain.
Project description:Laccases secreted by saprotrophic basidiomycete fungi are versatile biocatalysts able to oxidize a wide range of aromatic compounds using oxygen as the sole requirement. <i>Saccharomyces cerevisiae</i> is a preferred host for engineering fungal laccases. To assist the difficult secretion of active enzymes by yeast, the native signal peptide is usually replaced by the preproleader of <i>S. cerevisiae</i> alfa mating factor (MF?1). However, in most cases, only basal enzyme levels are obtained. During directed evolution in <i>S. cerevisiae</i> of laccases fused to the ?-factor preproleader, we demonstrated that mutations accumulated in the signal peptide notably raised enzyme secretion. Here we describe different protein engineering approaches carried out to enhance the laccase activity detected in the liquid extracts of <i>S. cerevisiae</i> cultures. We demonstrate the improved secretion of native and engineered laccases by using the fittest mutated ?-factor preproleader obtained through successive laccase evolution campaigns in our lab. Special attention is also paid to the role of protein N-glycosylation in laccase production and properties, and to the introduction of conserved amino acids through consensus design enabling the expression of certain laccases otherwise not produced by the yeast. Finally, we revise the contribution of mutations accumulated in laccase coding sequence (CDS) during previous directed evolution campaigns that facilitate enzyme production.
Project description:Laccases (EC 184.108.40.206) are enzymes known for their ability to catalyse the oxidation of phenolic compounds using molecular oxygen as the final electron acceptor. Lignin is a natural phenylpropanoids biopolymer whose degradation in nature is thought to be aided by enzymatic oxidation by laccases. Laccase activity is often measured spectrophotometrically on compounds such as syringaldazine and ABTS which poorly relate to lignin. We employed natural phenolic hydroxycinnamates having different degree of methoxylations, p-coumaric, ferulic and sinapic acid, and a lignin model OH-dilignol compound as substrates to assess enzyme kinetics by HPLC-MS on two fungal laccases Trametes versicolor laccase, Tv and Ganoderma lucidum laccase, Gl. The method allowed accurate kinetic measurements and detailed insight into the product profiles of both laccases. Both Tv and Gl laccase are active on the hydroxycinnammates and show a preference for substrate with methoxylations. Product profiles were dominated by the presence of dimeric and trimeric species already after 10 minutes of reaction and similar profiles were obtained with the two laccases. This new HPLC-MS method is highly suitable and accurate as a new method for assaying laccase activity on genuine phenolic substrates, as well as a tool for examining laccase oxidation product profiles.
Project description:Laccases are multicopper oxidases that are able to catalyze reactions involving a range of substrates, including phenols and amines, and this ability is related to the existence of different laccases. Basidiomycetes usually have more than one gene for laccase, but until now, this feature has not been demonstrated in a marine-derived fungus. Peniophora sp. CBMAI 1063 is a basidiomycete fungus isolated from a marine sponge that exhibits the ability to secrete significant amounts of laccase in saline conditions. In the present study, we identified laccase sequences from the transcriptome of Peniophora sp. CBMAI 1063 and used them to perform different molecular in silico analyses. The results revealed the presence of at least eight putative genes, which may encode ten different laccases with peptide lengths ranging from 482 to 588 aa and molecular weights ranging from 53.5 to 64.4 kDa. These laccases seem to perform extracellular activities, with the exception of one that may represent an intracellular laccase. The 10 predicted laccases expressed by Peniophora sp. CBMAI 1063 in laccase-induced media showed different patterns of N-glycosylation and isoelectric points and are divided into two classes based on the residue associated with the regulation of the redox potential of the enzyme. None of the predicted laccases showed more than 61% similarity to other fungal laccases. Based on the differences among the laccases expressed by Peniophora sp. CBMAI 1063, this marine-derived basidiomycete represents a valuable resource with strong potential for biotechnological exploitation.
Project description:Laccases belong to the group of phenol oxidizes and constitute one of the most promising classes of enzymes for future use in various fields. For industrial and biotechnological purposes, laccases were among the first enzymes providing larger-scale applications such as removal of polyphenols or conversion of toxic compounds. The wood-degrading basidiomycete Cerrena unicolor C-139, reported in this study, is one of the high-laccase producers. In order to facilitate novel and more efficient biocatalytic process applications, there is a need for laccases with improved biochemical properties, such as thermostability or stability in broad ranges of pH. In this work, modifications of laccase isoforms by hydrophobization, hydrophilization, and polymerization were performed. The hydrophobized and hydrophilized enzyme showed enhanced surface activity and higher ranges of pH and temperatures in comparison to its native form. However, performed modifications did not appear to noticeably alter enzyme's native structure possibly due to the formation of coating by particles of saccharides around the molecule. Additionally, surface charge of modified laccase shifted towards the negative charge for the hydrophobized laccase forms. In all tested modifications, the size exclusion method led to average 80 % inhibition removal for hydrophilized samples after an hour of incubation with fluoride ions. Samples that were hydrophilized with lactose and cellobiose showed an additional 90 % reversibility of inhibition by fluoride ions after an hour of concluding the reaction and 40 % after 24 h. The hydrophobized laccase showed higher level of the reversibility after 1 h (above 80 %) and 24 h (above 70 %) incubation with fluoride ions. The addition of ascorbate to laccase solution before a fluoride spike resulted in more efficient reversibility of fluoride inhibitory effect in comparison to the treatments with reagents used in the reversed sequence.
Project description:Laccases belong to the group of multicopper oxidases that exhibit wide substrate specificity for polyphenols and aromatic amines. They are found in plants, fungi, bacteria, and insects. In insects the only known role for laccase is in cuticle sclerotization. However, extracting laccase from the insect's cuticle requires proteolysis, resulting in an enzyme that is missing its amino-terminus. To circumvent this problem, we expressed and purified full-length and amino-terminally truncated recombinant forms of laccase-2 from the tobacco hornworm, Manduca sexta. We also purified the endogenous enzyme from the pharate pupal cuticle and used peptide mass fingerprinting analysis to confirm that it is laccase-2. All three enzymes had pH optima between 5 and 5.5 when using N-acetyldopamine (NADA) or N-beta-alanyldopamine-alanyldopamine (NBAD) as substrates. The laccases exhibited typical Michaelis-Menten kinetics when NADA was used as a substrate, with K(m) values of 0.46 mM, 0.43 mM, and 0.63 mM, respectively, for the full-length recombinant, truncated recombinant, and cuticular laccases; the apparent k(cat) values were 100 min(-1), 80 min(-1), and 290 min(-1). The similarity in activity of the two recombinant laccases suggests that laccase-2 is expressed in an active form rather than as a zymogen, as had been previously proposed. This conclusion is consistent with the detection of activity in untanned pupal wing cuticle using the laccase substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Immunoblot analysis of proteins extracted from both tanned and untanned cuticle detected only a single protein of 84 kDa, consistent with the full-length enzyme. With NBAD as substrate, the full-length recombinant and cuticular laccases showed kinetics indicative of substrate inhibition, with K(m) values of 1.9 mM and 0.47 mM, respectively, and apparent k(cat) values of 200 min(-1) and 180 min(-1). These results enhance our understanding of cuticle sclerotization, and may aid in the design of insecticides targeting insect laccases.