Date Published: May 24, 2018
Publisher: Public Library of Science
Author(s): Io Antonopoulou, Cameron Hunt, Gabriella Cerullo, Simona Varriale, Alexandra Gerogianni, Vincenza Faraco, Ulrika Rova, Paul Christakopoulos, Albert Jeltsch.
The type C feruloyl esterase FoFaeC from Fusarium oxysporum is a newly discovered enzyme with high potential for use in the hydrolysis of lignocellulosic biomass but it shows low activity towards sinapates. In this work, small molecule docking simulations were employed in order to identify important residues for the binding of the four model methyl esters of hydroxycinnamic acids, methyl ferulate/caffeate/sinapate/p-coumarate, to the predicted structure of FoFaeC. Subsequently rational redesign was applied to the enzyme’ active site in order to improve its specificity towards methyl sinapate. A double mutation (F230H/T202V) was considered to provide hydrophobic environment for stabilization of the methoxy substitution on sinapate and a larger binding pocket. Five mutant clones and the wild type were produced in Pichia pastoris and biochemically characterized. All clones showed improved activity, substrate affinity, catalytic efficiency and turnover rate compared to the wild type against methyl sinapate, with clone P13 showing a 5-fold improvement in catalytic efficiency. Although the affinity of all mutant clones was improved against the four model substrates, the catalytic efficiency and turnover rate decreased for the substrates containing a hydroxyl substitution.
Feruloyl esterases (EC 18.104.22.168, FAEs) are a subclass of carbohydrate esterases that are considered a biotechnological key for the degradation of lignocellulosic biomass, catalyzing the hydrolysis of the ester bond between hydroxycinnamic acids, such as ferulic acid (FA), caffeic acid (CA), sinapic acid (SA), p-coumaric acid (pCA) and sugars found in plant cell walls. Their application as accessory enzymes for hydrolysis as well as for the synthesis of bioactive compounds has been underlined during the past years [1–4]. A widely accepted system for the classification of FAEs is based on their specificity towards the hydrolysis of methyl esters of hydroxycinnamic acids: methyl ferulate (MFA), methyl caffeate (MCA), methyl sinapate (MSA) and methyl p-coumarate (MpCA) (Fig 1).Type A FAEs show preference on methoxy substituted substrates, such as MFA and MSA, are active on MpCA and diferulates but not MCA, while Type B FAEs show preference on hydroxy substituted substrates, MpCA and MCA, are active on MFA, but not active against MSA and diferulates. Type C and D have specificity towards all four substrates, but only type D FAEs are active towards diferulates [5–8]. Other classification systems for fungal FAEs have been based on phylogenetic analysis and functionality [9–12].
The rational redesign of the active site of type C FoFaeC provided an insight into the hydrolytic mechanisms of this enzyme and opens the way for a new approach on the exploitation of FAEs for use in novel bio catalytic processes by tailoring their specificity according to the desired reaction.