Exploring laccase-like multicopper oxidase genes from the ascomycete Trichoderma reesei: a functional, phylogenetic and evolutionary study

<p>Abstract</p> <p>Background</p> <p>The diversity and function of ligninolytic genes in soil-inhabiting ascomycetes has not yet been elucidated, despite their possible role in plant litter decay processes. Among ascomycetes, <it>Trichoderma reesei </it>is a model organism of cellulose and hemicellulose degradation, used for its unique secretion ability especially for cellulase production. <it>T. reesei </it>has only been reported as a cellulolytic and hemicellulolytic organism although genome annotation revealed 6 laccase-like multicopper oxidase (LMCO) genes. The purpose of this work was i) to validate the function of a candidate LMCO gene from <it>T. reesei</it>, and ii) to reconstruct LMCO phylogeny and perform evolutionary analysis testing for positive selection.</p> <p>Results</p> <p>After homologous overproduction of a candidate LMCO gene, extracellular laccase activity was detected when ABTS or SRG were used as substrates, and the recombinant protein was purified to homogeneity followed by biochemical characterization. The recombinant protein, called TrLAC1, has a molecular mass of 104 kDa. Optimal temperature and pH were respectively 40-45°C and 4, by using ABTS as substrate. TrLAC1 showed broad pH stability range of 3 to 7. Temperature stability revealed that TrLAC1 is not a thermostable enzyme, which was also confirmed by unfolding studies monitored by circular dichroism. Evolutionary studies were performed to shed light on the LMCO family, and the phylogenetic tree was reconstructed using maximum-likelihood method. LMCO and classical laccases were clearly divided into two distinct groups. Finally, Darwinian selection was tested, and the results showed that positive selection drove the evolution of sequences leading to well-known laccases involved in ligninolysis. Positively-selected sites were observed that could be used as targets for mutagenesis and functional studies between classical laccases and LMCO from <it>T. reesei</it>.</p> <p>Conclusions</p> <p>Homologous production and evolutionary studies of the first LMCO from the biomass-degrading fungus <it>T. reesei </it>gives new insights into the physicochemical parameters and biodiversity in this family.</p

Mechanisms of laccase-mediator treatments improving the enzymatic hydrolysis of pre-treated spruce

Abstract Background The recalcitrance of softwood to enzymatic hydrolysis is one of the major bottlenecks hindering its profitable use as a raw material for platform sugars. In softwood, the guaiacyl-type lignin is especially problematic, since it is known to bind hydrolytic enzymes non-specifically, rendering them inactive towards cellulose. One approach to improve hydrolysis yields is the modification of lignin and of cellulose structures by laccase-mediator treatments (LMTs). Results LMTs were studied to improve the hydrolysis of steam pre-treated spruce (SPS). Three mediators with three distinct reaction mechanisms (ABTS, HBT, and TEMPO) and one natural mediator (AS, that is, acetosyringone) were tested. Of the studied LMTs, laccase-ABTS treatment improved the degree of hydrolysis by 54%, while acetosyringone and TEMPO increased the hydrolysis yield by 49% and 36%, respectively. On the other hand, laccase-HBT treatment improved the degree of hydrolysis only by 22%, which was in the same order of magnitude as the increase induced by laccase treatment without added mediators (19%). The improvements were due to lignin modification that led to reduced adsorption of endoglucanase Cel5A and cellobiohydrolase Cel7A on lignin. TEMPO was the only mediator that modified cellulose structure by oxidizing hydroxyls at the C6 position to carbonyls and partially further to carboxyls. Oxidation of the reducing end C1 carbonyls was also observed. In contrast to lignin modification, oxidation of cellulose impaired enzymatic hydrolysis. Conclusions LMTs, in general, improved the enzymatic hydrolysis of SPS. The mechanism of the improvement was shown to be based on reduced adsorption of the main cellulases on SPS lignin rather than cellulose oxidation. In fact, at higher mediator concentrations the advantage of lignin modification in enzymatic saccharification was overcome by the negative effect of cellulose oxidation. For future applications, it would be beneficial to be able to understand and modify the binding properties of lignin in order to decrease unspecific enzyme binding and thus to increase the mobility, action, and recyclability of the hydrolytic enzymes

Leukotriene A4 hydrolase : Exploration of the active sites and catalytic mechanisms by site-directed mutagenesis

Leukotriene (LT) A4 is a pivotal intermediate in the biosynthesis of leukotrienes, a family of potent lipid mediators involved in a variety of inflammatory and allergic disorders. The bifunctional zinc metalloenzyme LTA4 hydrolase converts LTA4 into LTB4, one of the most potent chemotaxins known to date. In addition, LTA4 hydrolase possesses a peptide cleaving activity, the physiological role of which is presently unknown. From sequence comparisons with aminopeptidase M, Tyr-383 in LTA4 hydrolase was suggested as a potential catalytic residue. Tyr-383 was exchanged to a Phe, His or Gln by site-directed mutagenesis, and the purified recombinant enzymes were devoid of peptidase activity but displayed significant epoxide hydrolase activity. The results indicate that Tyr-383 is located at the active site, and suggests a role for Tyr-383 in the peptidase reaction where it may serve as a proton donor. Mutagenetic replacement of the conserved Glu-296 in LTA4 hydrolase with a Gln, selectively abrogates its peptidase activity. Similar results were obtained when Glu was exchanged for Asp or Asn. Thus, a carboxylate at a proper distance is required at position 296 for the enzyme to exhibit peptidase activity. The role of Glu-296 for the binding of bestatin, a prototype for an aminopeptidase inhibitor, was also studied. When using LTA4 as substrate, the ability of bestatin to inhibit the mutants dropped dramatically (< 0.7 % of the control), which indicates that Glu-296 is critical for binding of this inhibitor. During catalysis, LTA4 hydrolase is suicide inactivated by its lipid substrate LTA4. The inactivation occurs via an irreversible covalent binding of LTA4 to the enzyme. Using differential peptide mapping of unmodified and suicide inactivated enzyme, a 21-residue peptide fragment (K21) encompassing the residues 365-385 of human LTA4 hydrolase, was shown to be involved in the covalent binding of LTA4 and LTA4 methyl and ethyl ester, during suicide inactivation. The degree of inactivation of both the epoxide hydrolase and the peptidase activity correlated well with the degree of peptide modification, and the competitive inhibitor bestatin could prevent enzyme inactivation and modification of K21. A modified form of peptide K21 was isolated from enzyme inactivated with LTA4 ethyl ester. Edman degradation of this peptide revealed a gap in the sequence corresponding to Tyr-378 in LTA4 hydrolase, indicating that this is the site to which LTA4 binds during suicide inactivation. Tyr-378 was exchanged to a Phe or Gln by site-directed mutagenesis. Enzyme activity determinations of the purified mutated proteins indicated that Tyr-378 is not critical for catalyses. In fact, mutation of Tyr to Phe led to an enzyme with significantly increased turnover of LTA4. Notably, the mutated enzymes were not inactivated or covalently modified by treatment with the substrate LTA4. Moreover, the mutants were found to convert LTA4 not only to LTB4, but also into a second previously unknown enzymatic product structurally identified as 5(S),12(R) dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid, i.e. delta6-trans-delta8-cis-LTB4. Hence, Tyr-378 is a major structural determinant for suicide inactivation of LTA4 hydrolase and seems to assist in the formation of the correct double bond geometry in LTB4. Further characterization of the mutants in position 383 revealed that they exhibited a second enzymatic activity. In addition to LTB4, the mutants produced a novel metabolite with the structure 5(S),6(S)-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid. The kinetic parameters for the formation of 5(S),6(S)-DHETE were found to be similar to those obtained for the formation of LTB4. From the stereochemical configuration of the vicinal diol it was inferred that 5(S),6(S) DHETE is formed via an SNI mechanism involving a carbocation intermediate, which in turn indicates that the enzymatic conversion of LTA4 into LTB4 follows the same mechanism. A functional and structural relationship between LTA4 hydrolase and soluble xenobiotic epoxide hydrolase (sEH)was indicated by the fact that mutants of LTA4 hydrolase and sEH convert LTA4 into 5(S),6(S)-DHETE and its epimer at C6, respectively. ISBN 91-628-2535-

Three-dimensional structure of xylonolactonase from Caulobacter crescentus:A mononuclear iron enzyme of the 6-bladed β-propeller hydrolase family

Xylonolactonase Cc XylC from Caulobacter crescentus catalyzes the hydrolysis of the intramolecular ester bond of d‐xylonolactone. We have determined crystal structures of Cc XylC in complex with d‐xylonolactone isomer analogues d‐xylopyranose and (r)‐(+)‐4‐hydroxy‐2‐pyrrolidinone at high resolution. Cc XylC has a 6‐bladed β‐propeller architecture, which contains a central open channel having the active site at one end. According to our previous native mass spectrometry studies, Cc XylC is able to specifically bind Fe(2+). The crystal structures, presented here, revealed an active site bound metal ion with an octahedral binding geometry. The side chains of three amino acid residues, Glu18, Asn146, and Asp196, which participate in binding of metal ion are located in the same plane. The solved complex structures allowed suggesting a reaction mechanism for intramolecular ester bond hydrolysis in which the major contribution for catalysis arises from the carbonyl oxygen coordination of the xylonolactone substrate to the Fe(2+). The structure of Cc XylC was compared with eight other ester hydrolases of the β‐propeller hydrolase family. The previously published crystal structures of other β‐propeller hydrolases contain either Ca(2+), Mg(2+), or Zn(2+) and show clear similarities in ligand and metal ion binding geometries to that of Cc XylC. It would be interesting to reinvestigate the metal binding specificity of these enzymes and clarify whether they are also able to use Fe(2+) as a catalytic metal. This could further expand our understanding of utilization of Fe(2+) not only in oxidative enzymes but also in hydrolases