Structural and molecular insights into a bifunctional glycoside hydrolase 30 xylanase specific to glucuronoxylan.

Glycoside hydrolase (GH) 30 family xylanases are enzymes of biotechnological interest due to their capacity to degrade recalcitrant hemicelluloses, such as glucuronoxylan (GX). This study focuses on a subfamily 7 GH30, TtXyn30A from Thermothelomyces thermophilus, which acts on GX in an "endo" and "exo" mode, releasing methyl-glucuronic acid branched xylooligosaccharides (XOs) and xylobiose, respectively. The crystal structure of inactive TtXyn30A in complex with 23-(4-O-methyl-α-D-glucuronosyl)-xylotriose (UXX), along with biochemical analyses, corroborate the implication of E233, previously identified as alternative catalytic residue, in the hydrolysis of decorated xylan. At the -1 subsite, the xylose adopts a distorted conformation, indicative of the Michaelis complex of TtXyn30AEE with UXX trapped in the semi-functional active site. The most significant structural rearrangements upon substrate binding are observed at residues W127 and E233. The structures with neutral XOs, representing the "exo" function, clearly show the nonspecific binding at aglycon subsites, contrary to glycon sites, where the xylose molecules are accommodated via multiple interactions. Last, an unproductive ligand binding site is found at the interface between the catalytic and the secondary ß-domain which is present in all GH30 enzymes. These findings improve current understanding of the mechanism of bifunctional GH30s, with potential applications in the field of enzyme engineering.

Considerations Regarding Activity Determinants of Fungal Polyphenol Oxidases Based on Mutational and Structural Studies.

Polyphenol oxidases (PPOs) are an industrially relevant family of enzymes, being involved in the postharvest browning of fruits and vegetables, as well as in human melanogenesis. Their involvement lies in their ability to oxidize phenolic or polyphenolic compounds, which subsequently form pigments. The PPO family includes tyrosinases and catechol oxidases, which, in spite of their high structural similarity, exhibit different catalytic activities. Long-standing research efforts have not yet managed to decipher the structural determinants responsible for this differentiation, as every new theory is disproved by a more recent study. In the present work, we combined biochemical along with structural data in order to better understand the function of a previously characterized PPO from Thermothelomyces thermophila (TtPPO). The crystal structure of a TtPPO variant, determined at 1.55 Å resolution, represents the second known structure of an ascomycete PPO. Kinetic data for structure-guided mutants prove the implication of "gate" residue L306, residue HB1+1 (G292), and HB2+1 (Y296) in TtPPO function against various substrates. Our findings demonstrate the role of L306 in the accommodation of bulky substrates and show that residue HB1+1 is unlikely to determine monophenolase activity, as was suggested from previous studies.IMPORTANCE PPOs are enzymes of biotechnological interest. They have been extensively studied both biochemically and structurally, with a special focus on the plant-derived counterparts. Even so, explicit description of the molecular determinants of their substrate specificity is still pending. For ascomycete PPOs, only one crystal structure has been determined so far, thus limiting our knowledge on this tree branch of the family. In the present study, we report the second crystal structure of an ascomycete PPO. Combined with site-directed mutagenesis and biochemical studies, we depict the amino acids in the vicinity of the active site that affect enzyme activity and perform a detailed analysis on a variety of substrates. Our findings improve current understanding of structure-function relations of microbial PPOs, which is a prerequisite for the engineering of biocatalysts of desired properties.

Comparison of three seemingly similar lytic polysaccharide monooxygenases from Neurospora crassa suggests different roles in plant biomass degradation.

Many fungi produce multiple lytic polysaccharide monooxygenases (LPMOs) with seemingly similar functions, but the biological reason for this multiplicity remains unknown. To address this question, here we carried out comparative structural and functional characterizations of three cellulose-active C4-oxidizing family AA9 LPMOs from the fungus Neurospora crassa, NcLPMO9A (NCU02240), NcLPMO9C (NCU02916), and NcLPMO9D (NCU01050). We solved the three-dimensional structure of copper-bound NcLPMO9A at 1.6-Å resolution and found that NcLPMO9A and NcLPMO9C, containing a CBM1 carbohydrate-binding module, bind cellulose more strongly and were less susceptible to inactivation than NcLPMO9D, which lacks a CBM. All three LPMOs were active on tamarind xyloglucan and konjac glucomannan, generating similar products but clearly differing in activity levels. Importantly, in some cases, the addition of phosphoric acid-swollen cellulose (PASC) had a major effect on activity: NcLPMO9A was active on xyloglucan only in the presence of PASC, and PASC enhanced NcLPMO9D activity on glucomannan. Interestingly, the three enzymes also exhibited large differences in their interactions with enzymatic electron donors, which could reflect that they are optimized to act with different reducing partners. All three enzymes efficiently used H2O2 as a cosubstrate, yielding product profiles identical to those obtained in O2-driven reactions with PASC, xyloglucan, or glucomannan. Our results indicate that seemingly similar LPMOs act preferentially on different types of copolymeric substructures in the plant cell wall, possibly because these LPMOs are functionally adapted to distinct niches differing in the types of available reductants.

Interactions of a fungal lytic polysaccharide monooxygenase with ß-glucan substrates and cellobiose dehydrogenase.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds using molecular oxygen and an external electron donor. We have used NMR and isothermal titration calorimetry (ITC) to study the interactions of a broad-specificity fungal LPMO, NcLPMO9C, with various substrates and with cellobiose dehydrogenase (CDH), a known natural supplier of electrons. The NMR studies revealed interactions with cellohexaose that center around the copper site. NMR studies with xyloglucans, i.e., branched ß-glucans, showed an extended binding surface compared with cellohexaose, whereas ITC experiments showed slightly higher affinity and a different thermodynamic signature of binding. The ITC data also showed that although the copper ion alone hardly contributes to affinity, substrate binding is enhanced for metal-loaded enzymes that are supplied with cyanide, a mimic of O2 (-) Studies with CDH and its isolated heme b cytochrome domain unambiguously showed that the cytochrome domain of CDH interacts with the copper site of the LPMO and that substrate binding precludes interaction with CDH. Apart from providing insights into enzyme-substrate interactions in LPMOs, the present observations shed new light on possible mechanisms for electron supply during LPMO action.

Versatile Fungal Polyphenol Oxidase with Chlorophenol Bioremediation Potential: Characterization and Protein Engineering.

Polyphenol oxidases (PPOs) have been mostly associated with the undesirable postharvest browning in fruits and vegetables and have implications in human melanogenesis. Nonetheless, they are considered useful biocatalysts in the food, pharmaceutical, and cosmetic industries. The aim of the present work was to characterize a novel PPO and explore its potential as a bioremediation agent. A gene encoding an extracellular tyrosinase-like enzyme was amplified from the genome of Thermothelomyces thermophila and expressed in Pichia pastoris The recombinant enzyme (TtPPO) was purified and biochemically characterized. Its production reached 40 mg/liter, and it appeared to be a glycosylated and N-terminally processed protein. TtPPO showed broad substrate specificity, as it could oxidize 28/30 compounds tested, including polyphenols, substituted phenols, catechols, and methoxyphenols. Its optimum temperature was 65°C, with a half-life of 18.3 h at 50°C, while its optimum pH was 7.5. The homology model of TtPPO was constructed, and site-directed mutagenesis was performed in order to increase its activity on mono- and dichlorophenols (di-CPs). The G292N/Y296V variant of TtPPO 5.3-fold increased activity on 3,5-dichlorophenol (3,5-diCP) compared to the wild type.IMPORTANCE A novel fungal PPO was heterologously expressed and biochemically characterized. Construction of single and double mutants led to the generation of variants with altered specificity against CPs. Through this work, knowledge is gained regarding the effect of mutations on the substrate specificity of PPOs. This work also demonstrates that more potent biocatalysts for the bioremediation of harmful CPs can be developed by applying site-directed mutagenesis.

Structural and Functional Characterization of a Lytic Polysaccharide Monooxygenase with Broad Substrate Specificity.

The recently discovered lytic polysaccharide monooxygenases (LPMOs) carry out oxidative cleavage of polysaccharides and are of major importance for efficient processing of biomass. NcLPMO9C from Neurospora crassa acts both on cellulose and on non-cellulose ß-glucans, including cellodextrins and xyloglucan. The crystal structure of the catalytic domain of NcLPMO9C revealed an extended, highly polar substrate-binding surface well suited to interact with a variety of sugar substrates. The ability of NcLPMO9C to act on soluble substrates was exploited to study enzyme-substrate interactions. EPR studies demonstrated that the Cu(2+) center environment is altered upon substrate binding, whereas isothermal titration calorimetry studies revealed binding affinities in the low micromolar range for polymeric substrates that are due in part to the presence of a carbohydrate-binding module (CBM1). Importantly, the novel structure of NcLPMO9C enabled a comparative study, revealing that the oxidative regioselectivity of LPMO9s (C1, C4, or both) correlates with distinct structural features of the copper coordination sphere. In strictly C1-oxidizing LPMO9s, access to the solvent-facing axial coordination position is restricted by a conserved tyrosine residue, whereas access to this same position seems unrestricted in C4-oxidizing LPMO9s. LPMO9s known to produce a mixture of C1- and C4-oxidized products show an intermediate situation.

Structural and functional studies of a Fusarium oxysporum cutinase with polyethylene terephthalate modification potential.

BACKGROUND: Cutinases are serine hydrolases that degrade cutin, a polyester of fatty acids that is the main component of plant cuticle. These biocatalysts have recently attracted increased biotechnological interest due to their potential to modify and degrade polyethylene terephthalate (PET), as well as other synthetic polymers. METHODS: A cutinase from the mesophilic fungus Fusarium oxysporum, named FoCut5a, was expressed either in the cytoplasm or periplasm of Escherichia coli BL21. Its X-ray structure was determined to 1.9Å resolution using molecular replacement. The activity of the recombinant enzyme was tested on a variety of synthetic esters and polyester analogues. RESULTS: The highest production of recombinant FoCut5a was achieved using periplasmic expression at 16°C. Its crystal structure is highly similar to previously determined Fusarium solani cutinase structure. However, a more detailed comparison of the surface properties and amino acid interactions revealed differences with potential impact on the biochemical properties of the two enzymes. FoCut5a showed maximum activity at 40°C and pH 8.0, while it was active on three p-nitrophenyl synthetic esters of aliphatic acids (C(2), C(4), C(12)), with the highest catalytic efficiency for the hydrolysis of the butyl ester. The recombinant cutinase was also found capable of hydrolyzing PET model substrates and synthetic polymers. CONCLUSIONS: The present work is the first reported expression and crystal structure determination of a functional cutinase from the mesophilic fungus F. oxysporum with potential application in surface modification of PET synthetic polymers. GENERAL SIGNIFICANCE: FoCut5a could be used as a biocatalyst in industrial applications for the environmentally-friendly treatment of synthetic polymers.

A thermostable GH26 endo-ß-mannanase from Myceliophthora thermophila capable of enhancing lignocellulose degradation.

The endomannanase gene em26a from the thermophilic fungus Myceliophthora thermophila, belonging to the glycoside hydrolase family 26, was functionally expressed in the methylotrophic yeast Pichia pastoris. The putative endomannanase, dubbed MtMan26A, was purified to homogeneity (60 kDa) and subsequently characterized. The optimum pH and temperature for the enzymatic activity of MtMan26A were 6.0 and 60 °C, respectively. MtMan26A showed high specific activity against konjac glucomannan and carob galactomannan, while it also exhibited high thermal stability with a half-life of 14.4 h at 60 °C. Thermostability is of great importance, especially in industrial processes where harsh conditions are employed. With the aim of better understanding its structure-function relationships, a homology model of MtMan26A was constructed, based on the crystallographic structure of a close homologue. Finally, the addition of MtMan26A as a supplement to the commercial enzyme mixture Celluclast® 1.5 L and Novozyme® 188 resulted in enhanced enzymatic hydrolysis of pretreated beechwood sawdust, improving the release of total reducing sugars and glucose by 13 and 12 %, respectively.

Enzymatic synthesis of model substrates recognized by glucuronoyl esterases from Podospora anserina and Myceliophthora thermophila.

Glucuronoyl esterases (GEs) are recently discovered enzymes that are suggested to cleave the ester bond between lignin alcohols and xylan-bound 4-O-methyl-D-glucuronic acid. Although their potential use for enhanced enzymatic biomass degradation and synthesis of valuable chemicals renders them attractive research targets for biotechnological applications, the difficulty to purify natural fractions of lignin-carbohydrate complexes hampers the characterization of fungal GEs. In this work, we report the synthesis of three aryl alkyl or alkenyl D-glucuronate esters using lipase B from Candida antarctica (CALB) and their use to determine the kinetic parameters of two GEs, StGE2 from the thermophilic fungus Myceliophthora thermophila (syn. Sporotrichum thermophile) and PaGE1 from the coprophilous fungus Podospora anserina. PaGE1 was functionally expressed in the methylotrophic yeast Pichia pastoris under the transcriptional control of the alcohol oxidase (AOX1) promoter and purified to its homogeneity (63 kDa). The three D-glucuronate esters contain an aromatic UV-absorbing phenol group that facilitates the quantification of their enzymatic hydrolysis by HPLC. Both enzymes were able to hydrolyze the synthetic esters with a pronounced preference towards the cinnamyl-D-glucuronate ester. The experimental results were corroborated by computational docking of the synthesized substrate analogues. We show that the nature of the alcohol portion of the hydrolyzed ester influences the catalytic efficiency of the two GEs.

The role of CE16 exo-deacetylases in hemicellulolytic enzyme mixtures revealed by the biochemical and structural study of the novel TtCE16B esterase.

Acetyl esterases belonging to the carbohydrate esterase family 16 (CE16) is a growing group of enzymes, with exceptional diversity regarding substrate specificity and regioselectivity. However, further insight into the CE16 specificity is required for their efficient biotechnological exploitation. In this work, exo-deacetylase TtCE16B from Thermothelomyces thermophila was heterologously expressed and biochemically characterized. The esterase targets positions O-3 and O-4 of singly and doubly acetylated non-reducing-end xylopyranosyl residues, provided the presence of a free vicinal hydroxyl group at position O-4 and O-3, respectively. Crystal structure of TtCE16B, the first representative among the CE16 enzymes, in apo- and product-bound form, allowed the identification of residues forming the catalytic triad and oxyanion hole, as well as the structural elements related to the enzyme preference for oligomers. The role of TtCE16B in hemicellulose degradation was investigated on acetylated xylan from birchwood and pre-treated beechwood biomass. TtCE16B exhibited complementary activity to commercially available OCE6 acetylxylan esterase. Moreover, it showed synergistic effects with SrXyl43 ß-xylosidase. Overall, supplementation of xylan-targeting enzymatic mixtures with both TtCE16B and OCE6 esterases led to a 3-fold or 4-fold increase in xylose release, when using TmXyn10 and TtXyn30A xylanases respectively.

The structure of a novel glucuronoyl esterase from Myceliophthora thermophila gives new insights into its role as a potential biocatalyst.

The increasing demand for the development of efficient biocatalysts is a consequence of their broad industrial applications. Typical difficulties that are encountered during their exploitation in a variety of processes are interconnected with factors such as temperature, pH, product inhibitors etc. To eliminate these, research has been directed towards the identification of new enzymes that would comply with the required standards. To this end, the recently discovered glucuronoyl esterases (GEs) are an enigmatic family within the carbohydrate esterase (CE) family. Structures of the thermophilic StGE2 esterase from Myceliophthora thermophila (synonym Sporotrichum thermophile), a member of the CE15 family, and its S213A mutant were determined at 1.55 and 1.9 Å resolution, respectively. The first crystal structure of the S213A mutant in complex with a substrate analogue, methyl 4-O-methyl-ß-D-glucopyranuronate, was determined at 2.35 Å resolution. All of the three-dimensional protein structures have an α/ß-hydrolase fold with a three-layer αßα-sandwich architecture and a Rossmann topology and comprise one molecule per asymmetric unit. These are the first crystal structures of a thermophilic GE both in an unliganded form and bound to a substrate analogue, thus unravelling the organization of the catalytic triad residues and their neighbours lining the active site. The knowledge derived offers novel insights into the key structural elements that drive the hydrolysis of glucuronic acid esters.

Recalcitrant polysaccharide degradation by novel oxidative biocatalysts.

The classical hydrolytic mechanism for the degradation of plant polysaccharides by saprophytic microorganisms has been reconsidered after the recent landmark discovery of a new class of oxidases termed lytic polysaccharide monooxygenases (LPMOs). LPMOs are of increased biotechnological interest due to their implication in lignocellulosic biomass decomposition for the production of biofuels and high-value chemicals. They act on recalcitrant polysaccharides by a combination of hydrolytic and oxidative function, generating oxidized and non-oxidized chain ends. They are copper-dependent and require molecular oxygen and an external electron donor for their proper function. In this review, we present the recent findings concerning the mechanism of action of these oxidative enzymes and identify issues and questions to be addressed in the future.

The xylobiohydrolase activity of a GH30 xylanase on natively acetylated xylan may hold the key for the degradation of recalcitrant xylan.

Acetyl substitutions are common on the hemicellulosic structures of lignocellulose, which up until recently were known to inhibit xylanase activity. Emerging data, however, suggest that xylanases are able to accommodate acetyl side-groups within their catalytic site. In the present work, a fungal GH30 xylanase from Thermothelomyces thermophila, namely TtXyn30A, was shown to release acetylated xylobiose when acting on pretreated lignocellulosic substrate. The released disaccharides could be acetylated at the 2-OH, 3-OH or both positions of the non-reducing end xylose, but the existence of the acetylation on the reducing end cannot be excluded. The synergy of TtXyn30A with acetyl esterases indicates that particular subsites within its active site cannot tolerate acetylated xylopyranose residues. Molecular docking showed that acetyl group can be accommodated on the 2- or 3-OH position of the non-reducing end xylose, unlike the reducing-end xylose (subsite -1), where only 3-OH decoration can be accommodated. Such insight into the catalytic activity of TtXyn30A could contribute to a better understanding of its biological role and thus lead to a more sufficient biotechnological utilization.

Structure-function studies of a novel laccase-like multicopper oxidase from Thermothelomyces thermophila provide insights into its biological role.

Multicopper oxidases are promiscuous biocatalysts with great potential for the production of industrial compounds. This study is focused on the elucidation of the structure-function determinants of a novel laccase-like multicopper oxidase from the thermophilic fungus Thermothelomyces thermophila (TtLMCO1), which is capable of oxidizing both ascorbic acid and phenolic compounds and thus is functionally categorized between the ascorbate oxidases and fungal ascomycete laccases (asco-laccases). The crystal structure of TtLMCO1, determined using an AlphaFold2 model due to a lack of experimentally determined structures of close homologues, revealed a three-domain laccase with two copper sites, lacking the C-terminal plug observed in other asco-laccases. Analysis of solvent tunnels highlighted the amino acids that are crucial for proton transfer into the trinuclear copper site. Docking simulations showed that the ability of TtLMCO1 to oxidize ortho-substituted phenols stems from the movement of two polar amino acids at the hydrophilic side of the substrate-binding region, providing structural evidence for the promiscuity of this enzyme.

Crystal structure of the Fusarium oxysporum tannase-like feruloyl esterase FaeC in complex with p-coumaric acid provides insight into ligand binding.

Feruloyl esterases (FAEs) hydrolyze the ester bonds between hydroxycinnamic acids and arabinose residues of plant cell walls and exhibit considerable diversity in terms of substrate specificity. Here, we report the crystal structure of an FAE from Fusarium oxysporum (FoFaeC) at 1.7 Å resolution in complex with p-coumaric acid, which is the first ligand-bound structure of a tannase-like FAE. Our data reveal local conformational changes around the active site upon ligand binding, suggesting alternation between an active and a resting state of the enzyme. A swinging tyrosine residue appears to be gating the substrate binding pocket, while the lid domain of the protein exerts substrate specificity by means of a well-defined hydrophobic core that encases the phenyl moiety of the substrate.

The structure of a GH10 xylanase from Fusarium oxysporum reveals the presence of an extended loop on top of the catalytic cleft.

Xylanase enzymes have been the focus of considerable research in recent decades owing to their extensive use in a variety of biotechnological applications. Previous structural studies of a number of GH10 xylanases revealed that all GH10 family members have the (ß/α)(8)-barrel fold and their catalytic site is conserved. The structure of a new GH10 xylanase from Fusarium oxysporum (FoXyn10a) was determined at 1.94 Šresolution from crystals belonging to the tetragonal space group P4(1)2(1)2 with five molecules per asymmetric unit. Comparison of the structure of FoXyn10a with previously determined structures of GH10 family members indicated that most of the differences were located in the loop regions between the ordered secondary-structure elements of the barrel, as expected. However, alignment of FoXyn10a with sequence and structural homologues denoted an atypically long loop connecting strand ß6b and helix α6 that was only present in one other GH10 xylanase, the structure of which is not known. This structural feature may be of functional importance, with potential implications in the catalytic efficiency of the enzyme.

Expression, characterization and structural modelling of a feruloyl esterase from the thermophilic fungus Myceliophthora thermophila.

A ferulic acid esterase (FAE) from the thermophilic fungus Myceliophthora thermophila (synonym Sporotrichum thermophile), belonging to the carbohydrate esterase family 1 (CE-1), was functionally expressed in methylotrophic yeast Pichia pastoris. The putative FAE from the genomic DNA was successfully cloned in P. pastoris X-33 to confirm that the enzyme exhibits FAE activity. The recombinant FAE was purified to its homogeneity (39 kDa) and subsequently characterized using a series of model substrates including methyl esters of hydroxycinnamates, alkyl ferulates and monoferuloylated 4-nitrophenyl glycosides. The substrate specificity profiling reveals that the enzyme shows a preference for the hydrolysis of methyl caffeate and p-coumarate and a strong preference for the hydrolysis of n-butyl and iso-butyl ferulate. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose, whilst it was found capable of de-esterifying acetylated glucuronoxylans. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with an M3 xylanase from Trichoderma longibrachiatum (a maximum of 41% total FA released after 1 h incubation). Prediction of the secondary structure of MtFae1a was performed in the PSIPRED server whilst modelling the 3D structure was accomplished by the use of the HH 3D structure prediction server.

Unique features of the bifunctional GH30 from Thermothelomyces thermophila revealed by structural and mutational studies.

Fungal xylanases belonging to family GH30_7, initially categorized as endo-glucuronoxylanases, are now known to differ both in terms of substrate specificity, as well as mode of action. Recently, TtXyn30A, a GH30_7 xylanase from Thermothelomyces thermophila, was shown to possess dual activity, acting on the xylan backbone in both an endo- and an exo- manner. Here, in an effort to identify the structural characteristics that append these functional properties to the enzyme, we present the biochemical characterization of various TtXyn30A mutants as well as its crystal structure, alone, and in complex with the reaction product. An auxiliary catalytic amino acid has been identified, while it is also shown that glucuronic acid recognition is not mediated by a conserved arginine residue, as shown by previously determined GH30 structures.

Functional expression of a thermophilic glucuronyl esterase from Sporotrichum thermophile: identification of the nucleophilic serine.

A glucuronyl esterase (GE) from the thermophilic fungus Sporotrichum thermophile, belonging to the carbohydrate esterase family 15 (CE-15), was functionally expressed in the methylotrophic yeast Pichia pastoris. The putative GE gene ge2 from the genomic DNA was successfully cloned in frame with the sequence for the Saccharomyces cerevisiae alpha-factor secretion signal under the transcriptional control of the alcohol oxidase (AOX1) promoter and integrated in P. pastoris X-33 to confirm that the encoded enzyme StGE2 exhibits esterase activity. The enzyme was active on substrates containing glucuronic acid methyl ester, showing optimal activity at pH 7.0 and 55 degrees C. The esterase displayed broad pH range stability between 4-10 and temperature stability up to 50 degrees C, rendering StGE2 a strong candidate for future biotechnological applications that require robust biocatalysts. ClustalW alignment of StGE2 with characterized GEs and selected homologous sequences, members of CE-15 family, revealed a novel consensus sequence G-C-S-R-X-G that features the characteristic serine residue involved in the generally conserved catalytic mechanism of the esterase family. The putative serine has been mutated, and the corresponding enzyme has been expressed in P. pastoris to prove that the candidate nucleophilic residue is responsible for catalyzing the enzymatic reaction.

The crystal structure of a Fusarium oxysporum feruloyl esterase that belongs to the tannase family.

Feruloyl esterases are enzymes of industrial interest that catalyse the hydrolysis of the ester bond between hydroxycinnamic acids such as ferulic acid and sugars present in the plant cell wall. Although there are several structures of biochemically characterized feruloyl esterases available, the structural determinants of their substrate specificity are not yet fully understood. Here, we present the crystal structure of a feruloyl esterase from Fusarium oxysporum (FoFaeC) at 2.3 Å resolution. Similar to the two other tannase-like feruloyl esterases, FoFaeC features a large lid domain covering the active site with potential regulatory role and a disulphide bond that brings together the serine and histidine of the catalytic triad. Differences are mainly observed in the metal coordination site and the substrate binding pocket. ENZYMES: E.C.3.1.1.73. DATABASES: The sequence of FoFaeC has been deposited with UniProt with accession code A0A1D3S5H0_FUSOX and the atomic coordinates of the three-dimensional structure with Protein Data Bank, with PDB code: 6FAT.