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 β-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 Cu2+ 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 study of biocatalysts implicated in hemicellulose degradation

The aim of the present PhD thesis was the biochemical and structural study of biocatalysts implicated in plant cell wall degradation. Two enzymes that belong to family glycoside hydrolase (GH) family 61 of CAZy (Carbohydrate-Active enZYmes) database were characterized biochemically while structural studies were carried out on a xylanase and a feruloyl esterase. FoCel61, a Fusarium oxysporum GH61 and StCel61, a Sporotrichum thermophile GH61 were cloned and heterologously expressed in the methylotrophic yeast Pichia pastoris. Their ability to hydrolyze a variety of polysaccharide substrates was subsequently investigated. Synergism experiments were performed by combining these enzymes with common cellulases and the resulting findings were correlated to the composition of the hydrolyzed material. It was found that both enzymes had the ability to increase the degree of lignocellulose conversion when combined with other cellulases and that this enhancing effect was dependent on the lignin content of the substrate. The structural characterization of two hemicellulases derived from F. oxysporum, a GH10 xylanase (FoXyn10a) and a type C feruloyl esterase (FoFaeC) was performed by X-ray crystallography. Diffracting crystals of both enzymes were grown under a variety of conditions. The structure of FoXyn10a was solved by molecular replacement using an homologous Cellumonas fimi xylanase as a starting model. FoXyn10a folds in the classical (8 barrel (TIM barrel) while the most striking difference observed, upon comparison with related GH10 structures, is the presence of an elongated loop above the catalytic cleft with possible functional role.Σκοπός της παρούσας διδακτορικής διατριβής ήταν η μελέτη βιοκαταλυτών που εμπλέκονται στην αποικοδόμηση της φυτικής βιομάζας με τη βοήθεια εργαλείων μοριακής και δομικής βιολογίας. Πιο συγκεκριμένα, χαρακτηρίστηκαν βιοχημικά δύο πρωτεΐνες της οικογένειας 61 της βάσης δεδομένων CAZy (Carbohydrate-Active enZYmes Database) και μελετήθηκαν κρυσταλλογραφικά δύο ένζυμα η δράση των οποίων ήταν ήδη γνωστή, μία ξυλανάση και μία εστεράση του φερουλικού οξέος. Οι πρωτεΐνες της οικογενείας 61 που μελετήθηκαν προέρχονταν από τους μύκητες Fusarium oxysporum (FoCel61) και Sporotrichum thermophile (StCel61). Κλωνοποιήθηκαν και εκφράστηκαν μέσω της μεθυλότροφης ζύμης Pichia pastoris και χαρακτηρίστηκαν ως προς την ικανότητά τους να υδρολύουν μία ποικιλία σακχαρούχων υποστρωμάτων. Εξετάστηκε επίσης η συμβολή τους στην αποικοδόμηση της φυτικής βιομάζας παρουσία των κλασικών κυτταρινασών και συσχετίστηκε η παρατηρούμενη συνεργιστική δράση με τη σύσταση των χρησιμοποιούμενων υλικών. Βρέθηκε ότι τα ένζυμα αυτά έχουν την ικανότητα να αυξάνουν το βαθμό μετατροπής των λιγνινοκυτταρινούχων υποστρωμάτων όταν συνδυάζονται με τα γνωστά υδρολυτικά ένζυμα που χρησιμοποιούνται ευρέως στις διεργασίες αυτές και ότι η βελτίωση αυτή σχετίζεται με την περιεκτικότητα του υποστρώματος σε λιγνίνη. Η δομική μελέτη δύο ημικυτταρινασών που προέρχονται από το F. oxysporum, μίας ξυλανάσης της οικογένειας 10 (FoXyn10a) και μίας εστεράσης του φερουλικού οξέος τύπου C (FoFaeC) πραγματοποιήθηκε με κρυσταλλογραφία ακτίνων Χ. Κρύσταλλοι των δύο ενζύμων με ικανότητα περίθλασης των ακτίνων Χ αναπτύχθηκαν σε μία ποικιλία συνθηκών. Για τον προσδιορισμό της δομής της FoXyn10a εφαρμόστηκε η τεχνική της μοριακής αντικατάστασης χρησιμοποιώντας ως αρχικό μοντέλο τη δομή μίας ομόλογης ξυλανάσης από το Cellumonas fimi. Η καινούρια δομή ανέδειξε ότι η FoXyn10a υιοθετεί τη διαμόρφωση (β/α)8 barrel, μία αναδίπλωση κοινή σε όλα τα μέλη της οικογένειας 10, αλλά και την παρουσία ενός καινούριου βρόχου κοντά στην είσοδο του ενεργού κεντρού με πιθανό λειτουργικό ρόλο

Institutions and Semantic Networks

61 σ.Παρουσιάζονται η θεωρίας των institutions καθώς και η έννοια του σημασιολογικού δικτύου και αποδεικνύεται ότι τα σημασιολογικά δίκτυα σχηματίζουν ένα institution.We present the theory of institutions and the concept of semantic network and prove that semantic networks form an institution.Μαρία Μ. Δημαρόγκων

Structural and Molecular Study of Biocatalysts implicated in Hemicellulose Degradation

163 σ.Τα τοιχώματα των φυτικών κυττάρων αποτελούν την πιο άφθονη πηγή οργανικού άνθρακα στη γη και η αξιοποίησή τους σε βιοτεχνολογικές εφαρμογές έχει προσελκύσει τα τελευταία χρόνια έντονο ερευνητικό ενδιαφέρον. Τα ένζυμα που συμβάλλουν στην αποικοδόμηση της φυτικής βιομάζας παρουσιάζουν μεγάλο ενδιαφέρον λόγω της εφαρμογής τους σε πληθώρα διεργασιών όπως είναι η βιομηχανία χάρτου, η αρτοποιία, η υφαντουργία, η παραγωγή ζωωτροφών και απορρυπαντικών και η παραγωγή βιοαιθανόλης δεύτερης γενιάς, χρησιμοποιώντας ως πρώτη ύλη κυτταρινούχα φυτά, αγροτικά ή δασικά παραπροϊόντα ή ακόμα και απορρίματα. Ειδικότερα, οι ερευνητικές προσπάθειες των τελευταίων ετών έχουν στραφεί στη μείωση του ενζυμικού φορτίου και κατά συνέπεια του κόστους που απαιτείται για τη μετατροπή της βιομάζας σε ζυμώσιμα σάκχαρα ώστε να είναι εφικτή και συμφέρουσα η παραγωγή βιοαιθανόλης σε βιομηχανικό επίπεδο. Σκοπός της παρούσας διδακτορικής διατριβής ήταν η μελέτη βιοκαταλυτών που εμπλέκονται στην αποικοδόμηση της φυτικής βιομάζας με τη βοήθεια εργαλείων μοριακής και δομικής βιολογίας. Πιο συγκεκριμένα, χαρακτηρίστηκαν βιοχημικά δύο πρωτεΐνες της οικογένειας 61 της βάσης δεδομένων CAZy (Carbohydrate-Active enZYmes Database) και μελετήθηκαν κρυσταλλογραφικά δύο ένζυμα η δράση των οποίων ήταν ήδη γνωστή, μία ξυλανάση και μία εστεράση του φερουλικού οξέος. Αρχικά γίνεται μία ανασκόπηση της σύστασης του φυτικού κυτταρικού τοιχώματος και των ενζύμων που συμβάλλουν στην αποικοδόμησή του. Τα ένζυμα αυτά έχουν κατηγοριοποιηθεί σε οικογένειες ανάλογα με την αμινοξική τους αλληλουχία. Αναλύονται ειδικότερα οι ιδιότητες και τα χαρακτηριστικά των ενζύμων που ανήκουν στις οικογένειες των γλυκοζιδικών υδρολασών 61 και 10 της CAZy και που μελετήθηκαν στo πλαίσιo της παρούσας εργασίας. Παρουσιάζεται επίσης η μέθοδος της κρυσταλλογραφίας ακτίνων Χ ως εργαλείο για τη δομική μελέτη των μακρομορίων. Οι πρωτεΐνες της οικογενείας 61 που μελετήθηκαν προέρχονταν από τους μύκητες Fusarium oxysporum (FoCel61) και Sporotrichum thermophile (StCel61). Κλωνοποιήθηκαν και εκφράστηκαν μέσω της μεθυλότροφης ζύμης Pichia pastoris και χαρακτηρίστηκαν ως προς την ικανότητά τους να υδρολύουν μία ποικιλία σακχαρούχων υποστρωμάτων. Εξετάστηκε επίσης η συμβολή τους στην αποικοδόμηση της φυτικής βιομάζας παρουσία των κλασικών κυτταρινασών και συσχετίστηκε η παρατηρούμενη συνεργιστική δράση με τη σύσταση των χρησιμοποιούμενων υλικών. Βρέθηκε ότι τα ένζυμα αυτά έχουν την ικανότητα να αυξάνουν το βαθμό μετατροπής των λιγνινοκυτταρινούχων υποστρωμάτων όταν συνδυάζονται με τα γνωστά υδρολυτικά ένζυμα που χρησιμοποιούνται ευρέως στις διεργασίες αυτές και ότι η βελτίωση αυτή σχετίζεται με την περιεκτικότητα του υποστρώματος σε λιγνίνη. Η δομική μελέτη δύο ημικυτταρινασών που προέρχονται από το F. oxysporum, μίας ξυλανάσης της οικογένειας 10 (FoXyn10a) και μίας εστεράσης του φερουλικού οξέος τύπου C (FoFaeC) πραγματοποιήθηκε με κρυσταλλογραφία ακτίνων Χ. Κρύσταλλοι των δύο ενζύμων με ικανότητα περίθλασης των ακτίνων Χ αναπτύχθηκαν σε μία ποικιλία συνθηκών. Για τον προσδιορισμό της δομής της FoXyn10a εφαρμόστηκε η τεχνική της μοριακής αντικατάστασης χρησιμοποιώντας ως αρχικό μοντέλο τη δομή μίας ομόλογης ξυλανάσης από το Cellumonas fimi. Η καινούρια δομή ανέδειξε ότι η FoXyn10a υιοθετεί τη διαμόρφωση (β/α)8 barrel, μία αναδίπλωση κοινή σε όλα τα μέλη της οικογένειας 10, αλλά και την παρουσία ενός καινούριου βρόχου κοντά στην είσοδο του ενεργού κεντρού με πιθανό λειτουργικό ρόλο.Plant cell walls are the most abundant source of organic carbon on the planet and their exploitation in biotechnological applications has attracted significant research interest. The enzymes implicated in plant biomass degradation are employed in a variety of industrial processes, ranging from paper, bread-making, textile, animal feed and detergent industry to the production of second generation bioethanol from cellulosic plants, agricultural and forestry byproducts or waste. Recent research efforts have focused on the reduction of protein loading required for the breakdown of lignocellulose to fermentable sugars, as the enzymic cost is currently the main obstacle of bioethanol production at industrial scale. The aim of the present PhD thesis was the biochemical and structural study of biocatalysts implicated in plant cell wall degradation. Two enzymes that belong to family glycoside hydrolase (GH) family 61 of CAZy (Carbohydrate-Active enZYmes) database were characterized biochemically while structural studies were carried out on a xylanase and a feruloyl esterase. In the first part of the thesis, the plant cell wall composition as well as the enzymes involved in its degradation are described. The latter have been divided into different families based on their amino acid sequence. The properties and characteristics of enzymes that belong to the GH families 61 and 10 of CAZy database are presented in more detail. The basic principles of X-ray crystallography, employed for the structural study of macromolecules, are also outlined. FoCel61, a Fusarium oxysporum GH61 and StCel61, a Sporotrichum thermophile GH61 were cloned and heterologously expressed in the methylotrophic yeast Pichia pastoris. Their ability to hydrolyze a variety of polysaccharide substrates was subsequently investigated. Synergism experiments were performed by combining these enzymes with common cellulases and the resulting findings were correlated to the composition of the hydrolyzed material. It was found that both enzymes had the ability to increase the degree of lignocellulose conversion when combined with other cellulases and that this enhancing effect was dependent on the lignin content of the substrate. The structural characterization of two hemicellulases derived from F. oxysporum, a GH10 xylanase (FoXyn10a) and a type C feruloyl esterase (FoFaeC) was performed by X-ray crystallography. Diffracting crystals of both enzymes were grown under a variety of conditions. The structure of FoXyn10a was solved by molecular replacement using an homologous Cellumonas fimi xylanase as a starting model. FoXyn10a folds in the classical (8 barrel (TIM barrel) while the most striking difference observed, upon comparison with related GH10 structures, is the presence of an elongated loop above the catalytic cleft with possible functional role.Μαρία Β. Δημαρόγκων

CELLULOSE DEGRADATION BY OXIDATIVE ENZYMES

Enzymatic degradation of plant biomass has attracted intensive research interest for the production of economically viable biofuels. Here we present an overview of the recent findings on biocatalysts implicated in the oxidative cleavage of cellulose, including polysaccharide monooxygenases (PMOs or LPMOs which stands for lytic PMOs), cellobiose dehydrogenases (CDHs) and members of carbohydrate-binding module family 33 (CBM33). PMOs, a novel class of enzymes previously termed GH61s, boost the efficiency of common cellulases resulting in increased hydrolysis yields while lowering the protein loading needed. They act on the crystalline part of cellulose by generating oxidized and non-oxidized chain ends. An external electron donor is required for boosting the activity of PMOs. We discuss recent findings concerning their mechanism of action and identify issues and questions to be addressed in the future

A Middle-Aged Enzyme Still in Its Prime: Recent Advances in the Field of Cutinases

Cutinases are α/β hydrolases, and their role in nature is the degradation of cutin. Such enzymes are usually produced by phytopathogenic microorganisms in order to penetrate their hosts. The first focused studies on cutinases started around 50 years ago. Since then, numerous cutinases have been isolated and characterized, aiming at the elucidation of their structure⁻function relations. Our deeper understanding of cutinases determines the applications by which they could be utilized; from food processing and detergents, to ester synthesis and polymerizations. However, cutinases are mainly efficient in the degradation of polyesters, a natural function. Therefore, these enzymes have been successfully applied for the biodegradation of plastics, as well as for the delicate superficial hydrolysis of polymeric materials prior to their functionalization. Even though research on this family of enzymes essentially began five decades ago, they are still involved in many reports; novel enzymes are being discovered, and new fields of applications arise, leading to numerous related publications per year. Perhaps the future of cutinases lies in their evolved descendants, such as polyesterases, and particularly PETases. The present article reviews the biochemical and structural characteristics of cutinases and cutinase-like hydrolases, and their applications in the field of bioremediation and biocatalysis

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

Recombinant expression of thermostable processive MtEG5 endoglucanase and its synergism with MtLPMO from Myceliophthora thermophila during the hydrolysis of lignocellulosic substrates

Abstract Background Filamentous fungi are among the most powerful cellulolytic organisms in terrestrial ecosystems. To perform the degradation of lignocellulosic substrates, these microorganisms employ both hydrolytic and oxidative mechanisms that involve the secretion and synergism of a wide variety of enzymes. Interactions between these enzymes occur on the level of saccharification, i.e., the release of neutral and oxidized products, but sometimes also reflected in the substrate liquefaction. Although the synergism regarding the yield of neutral sugars has been extensively studied, further studies should focus on the oxidized sugars, as well as the effect of enzyme combinations on the viscosity properties of the substrates. Results In the present study, the heterologous expression of an endoglucanase (EG) and its combined activity together with a lytic polysaccharide monooxygenase (LPMO), both from the thermophilic fungus Myceliophthora thermophila, are described. The EG gene, belonging to the glycoside hydrolase family 5, was functionally expressed in the methylotrophic yeast Pichia pastoris. The produced MtEG5A (75 kDa) featured remarkable thermal stability and showed high specific activity on microcrystalline cellulose compared to CMC, which is indicative of its processivity properties. The enzyme was capable of releasing high amounts of cellobiose from wheat straw, birch, and spruce biomass. Addition of MtLPMO9 together with MtEG5A showed enhanced enzymatic hydrolysis yields against regenerated amorphous cellulose (PASC) by improving the release not only of the neutral but also of the oxidized sugars. Assessment of activity of MtEG5A on the reduction of viscosity of PASC and pretreated wheat straw using dynamic viscosity measurements revealed that the enzyme is able to perform liquefaction of the model substrate and the natural lignocellulosic material, while when added together with MtLPMO9, no further synergistic effect was observed. Conclusions The endoglucanase MtEG5A from the thermophilic fungus M. thermophila exhibited excellent properties that render it a suitable candidate for use in biotechnological applications. Its strong synergism with LPMO was reflected in sugars release, but not in substrate viscosity reduction. Based on the level of oxidative sugar formation, this is the first indication of synergy between LPMO and EG reported

Recombinant expression of thermostable processive MtEG5 endoglucanase and its synergism with MtLPMO from Myceliophthora thermophila during the hydrolysis of lignocellulosic substrates

Abstract Background Filamentous fungi are among the most powerful cellulolytic organisms in terrestrial ecosystems. To perform the degradation of lignocellulosic substrates, these microorganisms employ both hydrolytic and oxidative mechanisms that involve the secretion and synergism of a wide variety of enzymes. Interactions between these enzymes occur on the level of saccharification, i.e., the release of neutral and oxidized products, but sometimes also reflected in the substrate liquefaction. Although the synergism regarding the yield of neutral sugars has been extensively studied, further studies should focus on the oxidized sugars, as well as the effect of enzyme combinations on the viscosity properties of the substrates. Results In the present study, the heterologous expression of an endoglucanase (EG) and its combined activity together with a lytic polysaccharide monooxygenase (LPMO), both from the thermophilic fungus Myceliophthora thermophila, are described. The EG gene, belonging to the glycoside hydrolase family 5, was functionally expressed in the methylotrophic yeast Pichia pastoris. The produced MtEG5A (75 kDa) featured remarkable thermal stability and showed high specific activity on microcrystalline cellulose compared to CMC, which is indicative of its processivity properties. The enzyme was capable of releasing high amounts of cellobiose from wheat straw, birch, and spruce biomass. Addition of MtLPMO9 together with MtEG5A showed enhanced enzymatic hydrolysis yields against regenerated amorphous cellulose (PASC) by improving the release not only of the neutral but also of the oxidized sugars. Assessment of activity of MtEG5A on the reduction of viscosity of PASC and pretreated wheat straw using dynamic viscosity measurements revealed that the enzyme is able to perform liquefaction of the model substrate and the natural lignocellulosic material, while when added together with MtLPMO9, no further synergistic effect was observed. Conclusions The endoglucanase MtEG5A from the thermophilic fungus M. thermophila exhibited excellent properties that render it a suitable candidate for use in biotechnological applications. Its strong synergism with LPMO was reflected in sugars release, but not in substrate viscosity reduction. Based on the level of oxidative sugar formation, this is the first indication of synergy between LPMO and EG reported

Backbone and side-chain ¹H, ¹³C, and ¹⁵N chemical shift assignments for the apo-form of the lytic polysaccharide monooxygenase NcLPMO9C

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