Substrate Report for: Ferulic-acid

Alzheimer's Disease (AD) is one of the most important neurodegenerative diseases and it covers 60% of whole dementia cases. AD is a constantly progressing neurodegenerative disease as a result of the production of beta-amyloid (Abeta) protein and the accumulation of hyper-phosphorylated Tau protein; it causes breakages in the synaptic bonds and neuronal deaths to a large extent. Millions of people worldwide suffer from AD because there is no definitive drug for disease prevention, treatment or slowdown. Over the last decade, multiple target applications have been developed for AD treatments. These targets include Abeta accumulations, hyper-phosphorylated Tau proteins, mitochondrial dysfunction, and oxidative stress resulting in toxicity. Various natural or semisynthetic antioxidant formulations have been shown to protect brain cells from Abeta induced toxicity and provide promising potentials for AD treatment. Ferulic acid (FA), a high-capacity antioxidant molecule, is naturally synthesized from certain plants. FA has been shown to have different substantial biological properties, such as anticancer, antidiabetic, antimicrobial, anti-inflammatory, hepatoprotective, and cardioprotective actions, etc. Furthermore, FA exerted neuroprotection via preventing Abeta-fibril formation, acting as an anti-inflammatory agent, and inhibiting free radical generation and acetylcholinesterase (AChE) enzyme activity. In this review, we present key biological roles of FA and several FA derivatives in Abeta-induced neurotoxicity, protection against free radical attacks, and enzyme inhibitions and describe them as possible therapeutic agents for the treatment of AD.

Lauryl ferulate (LF) was synthesized through lipase-catalyzed esterification of ferulic acid (FA) with lauryl alcohol in a novel ionic liquid ([(EO)-3C-im][NTf2]), and its antibacterial activities was evaluated in vitro against three food-related bacteria. [(EO)-3C-im][NTf2] was first synthesized through incorporating alkyl ether moiety into the double imidazolium ring. [(EO)-3C-im][NTf2] containing hexane was found to be the most suitable for this reaction. The effects of various parameters were studied, and the maximum yield of LF (90.1%) was obtained in the optimum reaction conditions, in [(EO)-3C-im][NTf2]/hexane (VILs:Vhexane=1:1) system, 0.08mmol/mL of FA concentration, 50mg/mL Novozym 435, 60 degrees C. LF exhibited a stronger antibacterial activity against Gram-negative (25 mm) than Gram-positive (21.5-23.2 mm) bacteria. The lowest MIC value was seen for E. coli (1.25mM), followed by L. Monocytogenes (2.5mM) and S.aureus (5mM). The MBCs for L. Monocytogenes, S.aureus and E. coli were 10, 20 and 5mM.

The feruloyl esterase (FAE) gene EST1 from the basidiomycete Pleurotus sapidus was heterologously expressed in Escherichia coli and Pichia pastoris. Catalytically active recombinant Est1 was secreted using P. pastoris as a host. For expression in P. pastoris, the expression vector pPIC9K was applied. The EST1 gene was cloned with an N-terminal alpha-mating factor pre-pro sequence and expressed under the control of a methanol inducible alcohol oxidase 1 promotor. Est1 was purified to homogeneity using ion exchange and hydrophobic interaction chromatography. The recombinant Est1 showed optima at pH 5.0 and 50 degrees C, and released ferulic acid from saccharide esters and from the natural substrate destarched wheat bran. Substrate specificity profile and descriptor-based analysis demonstrated unique properties, showing that Est1 did not fit into the current FAE classification model. Transferuloylation synthesis of feruloyl-saccharide esters was proven for mono- and disaccharides.

Ferulic acid is a common constituent of the plant cell-wall matrix where it decorates and can crosslink mainly arabinoxylans to provide structural reinforcement. Microbial feruloyl esterases (FAEs) specialize in catalyzing hydrolysis of the ester bonds between phenolic acids and sugar residues in plant cell-wall polysaccharides such as arabinoxylan to release cinnamoyl compounds. Feruloyl esterases from lactic acid bacteria (LAB) have been highlighted as interesting enzymes for their potential applications in the food and pharmaceutical industries; however, there are few studies on the activity and structure of FAEs of LAB origin. Here, we report the crystal structure and biochemical characterization of a feruloyl esterase (LbFAE) from Lentilactobacillus buchneri, a LAB strain that has been used as a silage additive. The LbFAE structure was determined in the absence and presence of product (FA) and reveals a new type of homodimer association not previously observed for fungal or bacterial FAEs. The two subunits associate to restrict access to the active site such that only single FA chains attached to arabinoxylan can be accommodated, an arrangement that excludes access to FA cross-links between arabinoxylan chains. This narrow specificity is further corroborated by the observation that no FA dimers are produced, only FA, when feruloylated arabinoxylan is used as substrate. Docking of arabinofuranosyl-ferulate in the LbFAE structure highlights the restricted active site and lends further support to our hypothesis that LbFAE is specific for single FA side chains in arabinoxylan.

Alzheimer's Disease (AD) is one of the most important neurodegenerative diseases and it covers 60% of whole dementia cases. AD is a constantly progressing neurodegenerative disease as a result of the production of beta-amyloid (Abeta) protein and the accumulation of hyper-phosphorylated Tau protein; it causes breakages in the synaptic bonds and neuronal deaths to a large extent. Millions of people worldwide suffer from AD because there is no definitive drug for disease prevention, treatment or slowdown. Over the last decade, multiple target applications have been developed for AD treatments. These targets include Abeta accumulations, hyper-phosphorylated Tau proteins, mitochondrial dysfunction, and oxidative stress resulting in toxicity. Various natural or semisynthetic antioxidant formulations have been shown to protect brain cells from Abeta induced toxicity and provide promising potentials for AD treatment. Ferulic acid (FA), a high-capacity antioxidant molecule, is naturally synthesized from certain plants. FA has been shown to have different substantial biological properties, such as anticancer, antidiabetic, antimicrobial, anti-inflammatory, hepatoprotective, and cardioprotective actions, etc. Furthermore, FA exerted neuroprotection via preventing Abeta-fibril formation, acting as an anti-inflammatory agent, and inhibiting free radical generation and acetylcholinesterase (AChE) enzyme activity. In this review, we present key biological roles of FA and several FA derivatives in Abeta-induced neurotoxicity, protection against free radical attacks, and enzyme inhibitions and describe them as possible therapeutic agents for the treatment of AD.

Lauryl ferulate (LF) was synthesized through lipase-catalyzed esterification of ferulic acid (FA) with lauryl alcohol in a novel ionic liquid ([(EO)-3C-im][NTf2]), and its antibacterial activities was evaluated in vitro against three food-related bacteria. [(EO)-3C-im][NTf2] was first synthesized through incorporating alkyl ether moiety into the double imidazolium ring. [(EO)-3C-im][NTf2] containing hexane was found to be the most suitable for this reaction. The effects of various parameters were studied, and the maximum yield of LF (90.1%) was obtained in the optimum reaction conditions, in [(EO)-3C-im][NTf2]/hexane (VILs:Vhexane=1:1) system, 0.08mmol/mL of FA concentration, 50mg/mL Novozym 435, 60 degrees C. LF exhibited a stronger antibacterial activity against Gram-negative (25 mm) than Gram-positive (21.5-23.2 mm) bacteria. The lowest MIC value was seen for E. coli (1.25mM), followed by L. Monocytogenes (2.5mM) and S.aureus (5mM). The MBCs for L. Monocytogenes, S.aureus and E. coli were 10, 20 and 5mM.

Lignocellulosic biomass is a promising renewable resource; however, deconstruction of this material is still the rate-limiting step. Major obstacles in the biocatalytic turnover of lignocellulose are ester-linked decorations that prevent access to primary structural polysaccharides. Enzymes targeting these esters represent promising biotools for increasing bioconversion efficiency. Ruminant livestock are unique in their ability to degrade lignocellulose through the action of their gut microbiome. The anaerobic fungi (phylum Neocallimastigomycota) are key members of this ecosystem that express a large repertoire of carbohydrate-active enzymes (CAZymes) with little sequence identity with characterized CAZymes [Lombard, Golaconda, Drula, Coutinho and Henrissat (2014) Nucleic Acids Res. 42: , D490-D495]. We have identified a carbohydrate esterase family 1 (CE1) ferulic acid esterase (FAE) belonging toAnaeromyces mucronatus(AmCE1/Fae1a), and determined its X-ray structure in both the presence [1.55 A (1 A=0.1 nm)] and absence (1.60 A) of ferulic acid. AmCE1 adopts an alpha/beta-hydrolase fold that is structurally conserved with bacterial FAEs, and possesses a unique loop, termed the beta-clamp, that encloses the ligand. Isothermal titration calorimetry reveals that substrate binding is driven by enthalpic contributions, which overcomes a large entropic penalty. A comparative analysis of AmCE1 with related enzymes has uncovered the apparent structural basis for differential FAE activities targeting cross-linking ferulic acid conjugates compared with terminal decorations. Based on comparisons to structurally characterized FAEs, we propose that the beta-clamp may define the structural basis of exolytic activities in FAEs. This provides a structure-based tool for predicting exolysis and endolysis in CE1. These insights hold promise for rationally identifying enzymes tailored for bioconversion of biomass with variations in cell wall composition.

The feruloyl esterase (FAE) gene EST1 from the basidiomycete Pleurotus sapidus was heterologously expressed in Escherichia coli and Pichia pastoris. Catalytically active recombinant Est1 was secreted using P. pastoris as a host. For expression in P. pastoris, the expression vector pPIC9K was applied. The EST1 gene was cloned with an N-terminal alpha-mating factor pre-pro sequence and expressed under the control of a methanol inducible alcohol oxidase 1 promotor. Est1 was purified to homogeneity using ion exchange and hydrophobic interaction chromatography. The recombinant Est1 showed optima at pH 5.0 and 50 degrees C, and released ferulic acid from saccharide esters and from the natural substrate destarched wheat bran. Substrate specificity profile and descriptor-based analysis demonstrated unique properties, showing that Est1 did not fit into the current FAE classification model. Transferuloylation synthesis of feruloyl-saccharide esters was proven for mono- and disaccharides.

BACKGROUND: Microbial enzymes produced in the gastrointestinal tract are primarily responsible for the release and biochemical transformation of absorbable bioactive monophenols. In the present work we described the crystal structure of LJ0536, a serine cinnamoyl esterase produced by the probiotic bacterium Lactobacillus johnsonii N6.2. METHODOLOGY/PRINCIPAL FINDINGS: We crystallized LJ0536 in the apo form and in three substrate-bound complexes. The structure showed a canonical alpha/beta fold characteristic of esterases, and the enzyme is dimeric. Two classical serine esterase motifs (GlyXSerXGly) can be recognized from the amino acid sequence, and the structure revealed that the catalytic triad of the enzyme is formed by Ser(106), His(225), and Asp(197), while the other motif is non-functional. In all substrate-bound complexes, the aromatic acyl group of the ester compound was bound in the deepest part of the catalytic pocket. The binding pocket also contained an unoccupied area that could accommodate larger ligands. The structure revealed a prominent inserted alpha/beta subdomain of 54 amino acids, from which multiple contacts to the aromatic acyl groups of the substrates are made. Inserts of this size are seen in other esterases, but the secondary structure topology of this subdomain of LJ0536 is unique to this enzyme and its closest homolog (Est1E) in the Protein Databank. CONCLUSIONS: The binding mechanism characterized (involving the inserted alpha/beta subdomain) clearly differentiates LJ0536 from enzymes with similar activity of a fungal origin. The structural features herein described together with the activity profile of LJ0536 suggest that this enzyme should be clustered in a new group of bacterial cinnamoyl esterases.

Feruloyl esterases hydrolyse phenolic groups involved in the cross-linking of arabinoxylan to other polymeric structures. This is important for opening the cell wall structure making material more accessible to glycoside hydrolases. Here we describe the crystal structure of inactive S133A mutant of type-A feruloyl esterase from Aspergillus niger (AnFaeA) in complex with a feruloylated trisaccharide substrate. Only the ferulic acid moiety of the substrate is visible in the electron density map, showing interactions through its OH and OCH(3) groups with the hydroxyl groups of Tyr80. The importance of aromatic and polar residues in the activity of AnFaeA was also evaluated using site-directed mutagenesis. Four mutant proteins were heterologously expressed in Pichia pastoris, and their kinetic properties determined against methyl esters of ferulic, sinapic, caffeic and p-coumaric acid. The k(cat) of Y80S, Y80V, W260S and W260V was drastically reduced compared to that of the wild-type enzyme. However, the replacement of Tyr80 and Trp260 with smaller residues broadened the substrate specificity of the enzyme, allowing the hydrolysis of methyl caffeate. The role of Tyr80 and Trp260 in AnFaeA are discussed in light of the three-dimensional structure.

Feruloyl esterases function in the cleavage of ferulic acid's bonds to arabinoxylan and pectin where the ferulic acid moieties cross-link the layers of polysaccharide chains within hemicellulose. This work presents the crystal structure of FAE_XynZ, the domain of Clostridium thermocellum's cellulosomal xylanase Z that displays feruloyl esterase activity. The structure was obtained via multiple isomorphous replacement with anomalous scattering (MIRAS) using three heavy atom derivatives and refined against X-ray diffraction data of up to 1.75 A resolution. The R-value of the final model was 0.187 (R(free) = 0.21). FAE_XynZ displays an eight-stranded alpha/beta-fold with the characteristic "catalytic triad" at the heart of the active site. To define the substrate specificity determinants of the enzyme, the crystal structures of FAE_XynZ and the inactive FAE_XynZ(S172A) mutant were determined in complexes with the feruloyl-arabinoxylans FAXX and FAX(3), respectively. In the complex crystals, the ferulic acid moieties are clearly recognizable and allowed identification of the hydrophobic binding pocket. The carbohydrate part of both substrates is not visible in either structure. The location of the putative carbohydrate binding-pocket was inferred based on the location and orientation of the adjacent ferulic acid molecule. Five of the six residues lining the pocket were found to be conserved in FAE A from Orpinomyces sp., which further supports the proposed role of these amino acids.