Feruloyl Esterase Activity of the Clostridium thermocellum Cellulosome Can Be Attributed to Previously Unknown Domains of XynY and XynZ

ABSTRACT The cellulosome of Clostridium thermocellum is a multiprotein complex with endo- and exocellulase, xylanase, β-glucanase, and acetyl xylan esterase activities. XynY and XynZ, components of the cellulosome, are composed of several domains including xylanase domains and domains of unknown function (UDs). Database searches revealed that the C- and N-terminal UDs of XynY and XynZ, respectively, have sequence homology with the sequence of a feruloyl esterase of strain PC-2 of the anaerobic fungusOrpinomyces. Purified cellulosomes from C. thermocellum were found to hydrolyze FAXX (O-{5-O-[(E)-feruloyl]-α-l-arabinofuranosyl}-(1→3)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose) and FAX3(5-O-[(E)-feruloyl]-[O-β-d-xylopyranosyl-(1→2)]-O-α-l-arabinofuranosyl-[1→3]}-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose), yielding ferulic acid as a product, indicating that they have feruloyl esterase activity. Nucleotide sequences corresponding to the UDs of XynY and XynZ were cloned into Escherichia coli, and the expressed proteins hydrolyzed FAXX and FAX3. The recombinant feruloyl esterase domain of XynZ alone (FAEXynZ) and with the adjacent cellulose binding domain (FAE-CBDXynZ) were characterized. FAE-CBDXynZhad a molecular mass of 45 kDa that corresponded to the expected product of the 1,203-bp gene. Km andV max values for FAX3 were 5 mM and 12.5 U/mg, respectively, at pH 6.0 and 60°C. PAX3, a substrate similar to FAX3 but with ap-coumaroyl group instead of a feruloyl moiety was hydrolyzed at a rate 10 times slower. The recombinant enzyme was active between pH 3 to 10 with an optimum between pH 4 to 7 and at temperatures up to 70°C. Treatment of Coastal Bermuda grass with the enzyme released mainly ferulic acid and a lower amount ofp-coumaric acid. FAEXynZ had similar properties. Removal of the 40 C-terminal amino acids, residues 247 to 286, of FAEXynZ resulted in protein without activity. Feruloyl esterases are believed to aid in a release of lignin from hemicellulose and may be involved in lignin solubilization. The presence of feruloyl esterase in the C. thermocellumcellulosome together with its other hydrolytic activities demonstrates a powerful enzymatic potential of this organelle in plant cell wall decomposition.

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Regulation of the Feruloyl Esterase (faeA) Gene from Aspergillus niger

Applied and Environmental Microbiology ◽

10.1128/aem.65.12.5500-5503.1999 ◽

1999 ◽

Vol 65 (12) ◽

pp. 5500-5503 ◽

Cited By ~ 60

Author(s):

Ronald P. de Vries ◽

Jaap Visser

Keyword(s):

Cell Wall ◽

Aspergillus Niger ◽

Ferulic Acid ◽

Methoxy Group ◽

Plant Cell Wall ◽

Ring Structure ◽

Feruloyl Esterase ◽

Cell Wall Polysaccharides ◽

Aromatic Residues ◽

Encoding Gene

ABSTRACT Feruloyl esterases can remove aromatic residues (e.g., ferulic acid) from plant cell wall polysaccharides (xylan, pectin) and are essential for complete degradation of these polysaccharides. Expression of the feruloyl esterase-encoding gene (faeA) fromAspergillus niger depends on d-xylose (expression is mediated by XlnR, the xylanolytic transcriptional activator) and on a second system that responds to aromatic compounds with a defined ring structure, such as ferulic acid and vanillic acid. Several compounds were tested, and all of the inducing compounds contained a benzene ring which had a methoxy group at C-3 and a hydroxy group at C-4 but was not substituted at C-5. Various aliphatic groups occurred at C-1. faeA expression in the presence of xylose or ferulic acid was repressed by glucose. faeA expression in the presence of ferulic acid and xylose was greater thanfaeA expression in the presence of either compound alone. The various inducing systems allow A. niger to produce feruloyl esterase not only during growth on xylan but also during growth on other ferulic acid-containing cell wall polysaccharides, such as pectin.

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Enhancement of acetyl xylan esterase activity on cellulose acetate through fusion to a family 3 cellulose binding module

Enzyme and Microbial Technology ◽

10.1016/j.enzmictec.2015.07.001 ◽

2015 ◽

Vol 79-80 ◽

pp. 27-33 ◽

Cited By ~ 11

Author(s):

Galina Mai-Gisondi ◽

Ossi Turunen ◽

Ossi Pastinen ◽

Nikolaos Pahimanolis ◽

Emma R. Master

Keyword(s):

Cellulose Acetate ◽

Esterase Activity ◽

Acetyl Xylan Esterase ◽

Cellulose Binding Module ◽

Cellulose Binding

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Ferulic Acid Release and 4-Vinylguaiacol Formation during Brewing and Fermentation: Indications for Feruloyl Esterase Activity inSaccharomyces cerevisiae

Journal of Agricultural and Food Chemistry ◽

10.1021/jf0346556 ◽

2004 ◽

Vol 52 (3) ◽

pp. 602-608 ◽

Cited By ~ 136

Author(s):

Stefan Coghe ◽

Koen Benoot ◽

Filip Delvaux ◽

Bart Vanderhaegen ◽

Freddy R. Delvaux

Keyword(s):

Ferulic Acid ◽

Esterase Activity ◽

Feruloyl Esterase ◽

Acid Release

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Crystal Structure and Substrate Specificity Modification of Acetyl Xylan Esterase from Aspergillus luchuensis

Applied and Environmental Microbiology ◽

10.1128/aem.01251-17 ◽

2017 ◽

Vol 83 (20) ◽

Cited By ~ 8

Author(s):

Dai Komiya ◽

Akane Hori ◽

Takuya Ishida ◽

Kiyohiko Igarashi ◽

Masahiro Samejima ◽

...

Keyword(s):

Crystal Structure ◽

Cell Wall ◽

Substrate Specificity ◽

Ferulic Acid ◽

Plant Cell ◽

Plant Cell Wall ◽

Catalytic Triad ◽

Acetyl Xylan Esterase ◽

Content Type ◽

Aspergillus Luchuensis

ABSTRACT Acetyl xylan esterase (AXE) catalyzes the hydrolysis of the acetyl bonds present in plant cell wall polysaccharides. Here, we determined the crystal structure of AXE from Aspergillus luchuensis (AlAXEA), providing the three-dimensional structure of an enzyme in the Esterase_phb family. AlAXEA shares its core α/β-hydrolase fold structure with esterases in other families, but it has an extended central β-sheet at both its ends and an extra loop. Structural comparison with a ferulic acid esterase (FAE) from Aspergillus niger indicated that AlAXEA has a conserved catalytic machinery: a catalytic triad (Ser119, His259, and Asp202) and an oxyanion hole (Cys40 and Ser120). Near the catalytic triad of AlAXEA, two aromatic residues (Tyr39 and Trp160) form small pockets at both sides. Homology models of fungal FAEs in the same Esterase_phb family have wide pockets at the corresponding sites because they have residues with smaller side chains (Pro, Ser, and Gly). Mutants with site-directed mutations at Tyr39 showed a substrate specificity similar to that of the wild-type enzyme, whereas those with mutations at Trp160 acquired an expanded substrate specificity. Interestingly, the Trp160 mutants acquired weak but significant type B-like FAE activity. Moreover, the engineered enzymes exhibited ferulic acid-releasing activity from wheat arabinoxylan. IMPORTANCE Hemicelluloses in the plant cell wall are often decorated by acetyl and ferulic acid groups. Therefore, complete and efficient degradation of plant polysaccharides requires the enzymes for cleaving the side chains of the polymer. Since the Esterase_phb family contains a wide array of fungal FAEs and AXEs from fungi and bacteria, our study will provide a structural basis for the molecular mechanism of these industrially relevant enzymes in biopolymer degradation. The structure of the Esterase_phb family also provides information for bacterial polyhydroxyalkanoate depolymerases that are involved in biodegradation of thermoplastic polymers.

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Hydroxycinnamic acids and ferulic acid esterase in relation to biodegradation of complex plant cell walls

Canadian Journal of Animal Science ◽

10.4141/a04-010 ◽

2005 ◽

Vol 85 (3) ◽

pp. 255-267 ◽

Cited By ~ 20

Author(s):

P. Yu ◽

J. J. McKinnon ◽

D. A. Christensen

Keyword(s):

Cell Wall ◽

Ferulic Acid ◽

Plant Cell ◽

Cell Walls ◽

Plant Cell Wall ◽

Structural Characteristics ◽

Hydroxycinnamic Acid ◽

Feruloyl Esterase ◽

Plant Cell Walls ◽

Ferulic Acid Esterase

Ferulic acid (3-methoxy-4-hydroxycinnamic acid), present in complex plant cell walls, is covalently cross-linked to polysaccharides by ester bonds and to components of lignin mainly by ether bonds. Ferulic acid has also been shown to occur in dimer- and trimerized forms through oxidative coupling between esterified and/or etherified ferulic acid residues. These cross-links are among the factors most inhibitory to digestion of complex plant cell walls in ruminants. Recently obtained information on ferulic acid and ferulic acid esterases in relation to complex plant cell wall biodegradation is reviewed. A focus of the review is on structural characteristics of plant cell walls associated with ferulic acid, physicochemical properties of ferulic acid esterase and synergistic interaction between ferulic acid esterase and other accessary cell wall degrading enzymes on the release of ferulic acid and plant cell wall biodegradation. Key words: Ferulic acid, hydroxycinnamic acid, feruloyl esterase, interaction effects, polysaccharide, feruloyl-polysaccharides, plant cell walls, biodegradation

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Feruloyl Esterase Fae1 is Required Specifically for Host Colonisation by The Rice-Blast Fungus Magnaporthe Oryzae

10.21203/rs.3.rs-591209/v1 ◽

2021 ◽

Author(s):

Akhil Thaker ◽

Khyati Mehta ◽

Rajesh Patkar

Keyword(s):

Cell Wall ◽

Ferulic Acid ◽

Plant Cell ◽

Magnaporthe Oryzae ◽

Plant Cell Wall ◽

Biofuel Production ◽

Feruloyl Esterase ◽

Potential Candidate ◽

Blast Fungus ◽

Host Invasion

Abstract Plant cell wall acts as a primary barrier for microbial pathogens during infection. A cell wall degrading enzyme thus may be a crucial virulence factor, as it may aid the pathogen in successful host invasion. Nine genes coding for feruloyl esterases (Fae), likely involved in plant cell wall degradation, have been annotated in the genome of the cereal-blast fungus Magnaporthe oryzae . However, role of any Fae in pathogenicity of M. oryzae remains hitherto under explored. Here, we identified FAE1 gene (MGG_08737) that was significantly upregulated during host penetration and subsequent colonisation stages of infection. Accordingly, while deletion of FAE1 in M. oryzae did not affect the vegetative growth and asexual development, the fae1Δ mutant showed significantly reduced pathogenesis on rice plants, mainly due to impaired host invasion and colonisation. Very few (<10%) fae1Δ appressoria that formed the primary invasive hyphae, failed to elaborate from the first invaded cell to the neighboring plant cells. Interestingly, exogenously added glucose, as a simple carbon source, or ferulic acid, a product of the Fae activity, significantly supported the invasive growth of the fae1Δ mutant. We show that the Fae1-based feruloyl esterase activity, by targeting the plant cell wall, plays an important role in accumulating ferulic acid and/or sugar molecules, as a likely energy source, to enable host invasion and colonisation by M. oryzae. Given its role in plant cell wall digestion and host colonisation, M. oryzae Fae1 could be a potential candidate for a novel antifungal strategy and a biotechnological application in biofuel production.

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Homologous xylanases from Clostridium thermocellum: evidence for bi-functional activity, synergism between xylanase catalytic modules and the presence of xylan-binding domains in enzyme complexes

Biochemical Journal ◽

10.1042/bj3420105 ◽

1999 ◽

Vol 342 (1) ◽

pp. 105-110 ◽

Cited By ~ 37

Author(s):

Ana C. FERNANDES ◽

Carlos M. G. A. FONTES ◽

Harry J. GILBERT ◽

Geoffrey P. HAZLEWOOD ◽

Tito H. FERNANDES ◽

...

Keyword(s):

Clostridium Thermocellum ◽

Plant Cell Wall ◽

Catalytic Domain ◽

Glycosyl Hydrolase ◽

Biochemical Properties ◽

Binding Domain ◽

Binding Domains ◽

A Cell ◽

Cellulose Binding ◽

Hydrolysis Of

Clostridium thermocellum produces a consortium of plant-cell-wall hydrolases that form a cell-bound multi-enzyme complex called the cellulosome. In the present study two similar xylanase genes, xynU and xynV, were cloned from C. thermocellum strain YS and sequenced. The deduced primary structures of both xylanases, xylanase U (XylU) and xylanase V (XylV), were homologous with the previously characterized xylanases from C. thermocellum strain F1. Truncated derivatives of XylV were produced and their biochemical properties were characterized. The xylanases were shown to be remarkably thermostable and resistant to proteolytic inactivation. The catalytic domains hydrolysed xylan by a typical endo-mode of action. The type VI cellulose-binding domain (CBD) homologue of XylV bound xylan and, to a smaller extent, Avicel and acid-swollen cellulose. Deletion of the CBD from XylV abolished the capacity of the enzymes to bind polysaccharides. The polysaccharide-binding domain was shown to have a key role in the hydrolysis of insoluble substrates by XylV. The C-terminal domain of XylV, which is absent from XylU, removed acetyl groups from acetylated xylan and acted in synergy with the glycosyl hydrolase catalytic domain of the enzyme to elicit the hydrolysis of acetylated xylan.

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