Abstract
Ferulic acid esterases (FAE, EC 3.1.1.73) cleave the arabinose hydroxycinnamate ester in plant hemicellulose and other related substrates. FAE are commonly categorised as type A-D based on catalytic activities towards model, short alkyl chain esters of hydroxycinnamates. However, this system correlates poorly with sequence and structural features of the enzymes. In this study, we investigated the basis of the type A categorisation of an FAE from Aspergillus niger, AnFaeA, by comparing its activity toward methyl and arabinose hydroxycinnamate esters. kcat/Km ratios revealed that AnFaeA hydrolysed arabinose ferulate 1600-fold, and arabinose caffeate 6.5 times more efficiently than their methyl ester counterparts. Furthermore, small docking studies showed that while all substrates adopted a catalytic orientation with requisite proximity to the catalytic serine, methyl caffeate and methyl p-coumarate preferentially formed alternative non-catalytic conformations that were energetically favoured. Arabinose ferulate was unable to adopt the alternative conformation while arabinose caffeate preferred the catalytic orientation. This study demonstrates that use of short alkyl chain hydroxycinnnamate esters can result in activity misclassification. The findings of this study provide a basis for developing a robust classification system for FAE and form the basis of sequence-function relationships for this class.
Highlights
Ferulic acid esterases (FAE, EC 3.1.1.73) cleave the ester linkage between hydroxycinnamate and the C(O)[5] position of an arabinose residue in hemicellulose, a plant-derived sugar polymer
The most common hydroxycinnamic acids found in plant biomass are ferulic, p-coumaric, caffeic and sinapinic[5] which vary in the number of hydroxy and/or methoxy substituents in the meta-positions of the aromatic ring (Fig. 1)
One of the leading classification systems for FAE is based on the ability of a candidate to hydrolyse model substrates, viz the methyl- or ethyl esters of hydroxycinnamic acids such as ferulate (MFA/EFA), methyl p-coumarate (MpCA), methyl caffeate (MCA) and methyl sinapate (MSA)
Summary
Kelle et al.[16,17] compared with model substrates. with both molecular modelling tools and more authentic enzyme substrates available, alternative approaches to FAE classification can be explored. We compare the catalytic activities of the type A FAE from A. niger, AnFaeA, toward the model hydroxycinnamic acid substrates, methyl ferulate and -caffeate, and their arabinose ester counterparts. The second most relevant enzyme to the A-D classification, AnFaeB from A. niger, which was initially categorized as type B has since been re-categorised as belonging to in a family of type C FAE due to its sequence similarity[11] This goes to further highlight the complexity of the current classification systems based on enzymatic activity. The Protein Database (PDB) contains only 10 records under EC of 3.1.1.73 (feruloyl esterase) with only approximately three other listed outside this set Of these AnFaeA has the most significant volume of literature relating to activity with a clear distinction of no activity on methyl caffeate. The findings of this study will aid in developing a robust classification system for FAE and form the basis of a structure-function relationship for this enzyme class, whereby features of FAE sequence, structure and activity are aligned
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
