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- Materials and methods
A cinnamoyl esterase, ferulic acid esterase A, from Aspergillus niger releases ferulic acid and 5-5- and 8-O-4-dehydrodiferulic acids from plant cell walls. The breakage of one or both ester bonds from dehydrodimer cross-links between plant cell wall polymers is essential for optimal action of carbohydrases on these substrates, but it is not known if cinnamoyl esterases can break these cross-links by cleaving one of the ester linkages which would not release the free dimer. It is difficult to determine the mechanism of the reaction on complex substrates, and so we have examined the catalytic properties of ferulic acid esterase A from Aspergillus niger using a range of synthetic ethyl esterified dehydrodimers (5-5-, 8-5-benzofuran and 8-O-4-) and two 5-5-diferulate oligosaccharides. Our results show that the esterase is able to cleave the three major dehydrodiferulate cross-links present in plant cell walls. The enzyme is highly specific at hydrolysing the 5-5- and the 8-5-benzofuran diferulates but the 8-O-4-is a poorer substrate. The hydrolysis of dehydrodiferulates to free acids occurs in two discrete steps, one involving dissociation of a monoesterified intermediate which is negatively charged at the pH of the reaction. Although ferulic acid esterase A was able to release monoesters as products of reactions with all three forms of diesters, only the 5-5- and the 8-O-4-monoesters were substrates for the enzyme, forming the corresponding free diferulic acids. The esterase cannot hydrolyse the second ester bond from the 8-5-benzofuran monoester and therefore, ferulic acid esterase A does not form 8-5-benzofuran diferulic acid. Therefore, ferulic acid esterase A from Aspergillus niger contributes to total plant cell wall degradation by cleaving at least one ester bond from the diferulate cross-links that exist between wall polymers but does not always release the free acid product.
Cinnamoyl esterases, a subclass of the carboxylic ester hydrolases (EC 188.8.131.52), hydrolyse the ester bond between hydroxycinnamic acids and sugars present in plant cell walls and have been purified and characterized from many microorganisms . They act synergistically with xylanases and pectinases to digest plant cell walls and facilitate the access of hydrolases to the backbone of wall polymers . Ferulic acid esterase A (FAEA) from Aspergillus niger is able to cleave specifically the (1→5) ester bond between ferulic acid and arabinose and shows high specificity of hydrolysis for a range of synthetic methyl esters of phenyl alkanoic acids . The rate of reaction increases markedly when the substrates are small soluble feruloylated oligosaccharides derived from plant cell walls [5,6]. The rate of catalysis and apparent affinity of FAEA are influenced by the substituents on the aromatic ring and by the type, position of attachment and number of sugar moieties . Although the catalytic properties of FAEA on a broad range of substrates have been well investigated, the topology of the active site has not yet been described. Based on a deduced sequence for FAEA  and chemical modification studies (F. O. Aliwan and G. Williamson, unpublished results), it appears that the enzyme active site contains a nucleophilic serine residue, and that a tryptophan residue and carboxylic groups are involved in substrate binding.
Peroxidase/H2O2 mediated oxidation of trans-ferulic acid esterified to the main polymers in primary plant cell walls can form several ferulic acid dehydrodimers (8-5-, 8-O-4-, 5-5- and 8-8-) , which have now been identified and quantified in several plant cell walls [8–12]. Ferulate dimers have been shown to covalently cross-link cell wall polymers [13–19] influencing physical and mechanical properties of the plant cell wall [20,21] and contributing to cell wall indigestibility, probably by limiting the accessibility of main chain-degrading enzymes to the structural polysaccharides [22,23].
It has been shown that some microbial esterases are able to release diferulic acids from plant cell walls. FAEA from A. niger is able to release 5-5-diferulic acid from presolubilized barley, wheat bran and sugar beet pulp [24,25]. The activity is increased in combination with xylanases. The esterase is also able to release some 8-O-4-diferulic acid from pretreated wheat bran . We hypothesize that hydrolysis of dehydrodiferulates by esterases could occur either through a single association of enzyme and substrate where both ester bonds in the diester are hydrolysed before the enzyme and free acid product dissociate (Scheme 1):
E + DS ⇄ E−DS → E−DS′ → E−MS → E−MS′ → Ε−diFA → Ε + diFA
or in two discrete steps where the monoester product of the first reaction is released and becomes a substrate for a second reaction (Scheme 2):
E + DS ⇄ E−DS → E−DS′ → E + MS → E−MS → E−MS′ → Ε + diFA
where E is the free enzyme, DS is the dehydrodimer diester substrate, MS is the dehydrodimer monoester product, E–DS and E–MS are the dehydrodimer diester and dehydrodimer monoester enzyme-substrate complexes, E–DS′ and E–MS′ are the corresponding acyl-enzymes and diFA is the free acid product, dehydrodiferulic acid.
The release of dimers by esterases has been studied in large complex substrates such as whole and solubilized plant cell walls, which do not allow for the estimation of kinetic constants to determine the specificity of the enzyme. The development of chemical and enzymic procedures to synthesize methyl and ethyl esterified dehydrodiferulates [8,26] has provided us with a new tool to examine the specificity of esterases for hydrolysing dehydrodiferulates present in plant cell walls. In this paper, we have determined the catalytic properties of the FAEA from A. niger on a range of synthetic ethyl diferulate esters (5-5-, 8-O-4- and 8-5-benzofuran) and two oligosaccharide 5-5-diferulates isolated from maize bran in order to enhance our understanding of the catalytic preferences of FAEA. We have also investigated whether the hydrolysis of both ester bonds from the diester substrates occurs through one or two separate reactions. On the basis of our results, we propose that FAEA from A. niger contributes to the total degradation of plant cell walls by cleaving the diferulic cross-links between cell wall polymers.
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- Materials and methods
FAEA from A. niger is able to hydrolyse either of the ester bonds in 5-5- and 8-O-4-dehydrodiferulates to form monoesters, and subsequently to hydrolyse the monoesters to form the corresponding free diferulic acids. The enzyme is also able to specifically cleave one of the ester bonds in 8-5-benzofuran diester forming one of the possible monoesters. These results are in agreement with the ability of the esterase to release 5-5- and 8-O-4-diferulic acids, but not 8-5-benzofuran diferulic acid, from solubilized plant cell wall materials [24,25].
The differences observed in the specificity of FAEA for the three types of dehydrodimer can be attributed to differences in their structure. It has been shown that the specificity of microbial esterases differs markedly across a series of synthetic substrates. The distance between the phenolic ring and the ester bond and the number and position of methoxy and hydroxy substitutions on the benzene ring are critical in determining the catalytic efficiency of FAEA . The inability of FAEA to hydrolyse the ester bond on ring A of the 8-5-benzofuran dimer may be due to the 7-O-4 ether linkage which alters the olefinic part of the molecule. This may affect the distance between this ester bond and the aromatic ring A, and hence prevent catalysis. Differences in conformation, intramolecular bond lengths and angles, and exposure of reactive groups and of ester bonds for each particular type of coupled ferulate have a clear effect on esterase activity.
We have shown that FAEA is highly efficient at hydrolysing diesterified dehydrodiferulates. The first product of the hydrolysis, always a monoester, is also a substrate for FAEA but the specificity is lower than for the diester, i.e. the kinetic data indicate that the enzyme in vivo is more efficient at breaking the cross-links between polymers than at releasing free diferulic acid. Therefore, release of diferulic acids from plant cell wall materials is not a good indicator of cross-link cleavage. In order to study the breaking of cross-links in plant cell walls, novel methods need to be developed, possibly involving in situ detection of dehydrodimers and putative monoester products in intact plant cell walls.
It has been shown that diferulate cross-links between the main polymers in plant cell walls reduce cell wall digestibility by limiting the accessibility of hydrolytic enzymes [22,23]. We propose that FAEA from A. niger contributes to plant cell wall degradation by cleaving the most abundant diferulate cross-links present in plant cell walls. It should be noted that FAEA exhibits highest specificity for hydrolysis of the 5-5-dimer which is not one of the most abundant dimers present in plant cell wall materials [8–12], but it is the only dehydrodimer for which there is direct evidence of involvement in cross-linking plant cell wall polysaccharides . These results appear to indicate that A. niger preferentially cleaves 5-5-cross-links as an important event in degradation of complex cell wall polymers. However, it is more likely that there are other esterases whose specificities complement those of A. niger FAEA in breaking these cross-links, and which form part of the battery of hydrolytic enzymes required for complete degradation of complex polysaccharides.
Structural studies and the use of specific chemical inhibitors have indicated that hydrophobic binding sites are important components of the active sites in several esterases with diverse specificities (e.g. acetylcholinesterases, butyrylcholinesterases, lipases, cholesterol esterases, carboxyl esterases, proteases). For example, the crystal structure of acetylcholinesterase reveals that the active site of the enzyme lies near the bottom of a narrow gorge which contains 14 aromatic residues . More recently it has been shown that a carboxylesterase from Pseudomonas fluorescens has an active-site cleft with an inside surface of the surrounding walls composed of mainly aliphatic side chains . Chemical modification studies (F. O. Aliwan and G. Williamson, unpublished results) suggest that a single tryptophan residue and carboxylic group(s) are involved in the binding of substrate by FAEA. Our data appear to indicate that FAEA binds substrates more tightly at lower pH. There was some evidence that for the monoester substrate, neutralization of the negative charge on the carboxylic group (at low pH) further improved the interaction (based on Km values) between the enzyme and the substrate. This may indicate that hydrophobic and/or carboxylic residues in FAEA are involved in catalysis, but, as Km values are a function of all the rate constants for all the steps on the reaction pathway, we cannot discount the effect of pH on these rate constants.
The presence of organic solvent in the reaction medium influences the interaction between enzyme and substrate, which is due mainly to electrostatic and hydrophobic interactions. In the case of hydrophobic binding, the Km value is usually higher in water-solvent mixtures compared to aqueous solutions . The fivefold increase in the Km of FAEA for ethyl ferulate in 20% dimethylsulfoxide suggests that hydrophobic interactions are involved in binding. As 5-5-, 8-O-4- and 8-5-benzofuran diferulic acids are more hydrophobic molecules than ferulic acid , the hydrophobicity of diferulate substrates probably also contributes to give the observed increase in specificity compared to ferulates.
The apparent affinity of feruloyl esterases for cinnamate substrates is greatly enhanced when the cinnamic acid is esterified to sugars compared to alkyl esters . The type of sugars present, the length of the oligosaccharide and the position of the linkage between the primary and secondary sugars, all influence the release of ferulic acid by FAEA . FAEA is more specific for 5-5-diferulate substrates derived from plant cell walls than for alkyl esters such as methyl and ethyl ferulate. The enzyme shows some specificity also for the oligosaccharide chain to which the 5-5 diferulate is linked. It was possible that for alkyl diester substrates, the release of monoester intermediates was due to a reduced affinity between FAEA and intermediate, whereas with sugar diesters, we may have expected the increased affinity to prevent dissociation of the enzyme–sugar monoester intermediate (Scheme 1). However, we have detected sugar monoesterified intermediates as a result of hydrolysis of sugar diester substrates, and this indicates that the presence of sugars does not prevent dissociation of the intermediate from the enzyme–substrate complex.
In conclusion, the hydrolysis of diferulates to free acids by FAEA from A. niger proceeds in two discrete steps involving dissociation of a monoester intermediate which is negatively charged at the pH of the reaction shown in Scheme 2.
The esterase is highly specific for hydrolysing one ester bond from 5-5- and 8-5-dehydrodiferulates which cross-link plant cell wall polymers and we propose that this esterase contributes to total degradation of plant cell walls by cleaving diferulate cross-links. Our results also provide some evidence for the involvement of carboxylic groups and hydrophobic interactions in the binding of substrates.