Toggle Main Menu Toggle Search

Open Access padlockePrints

A modular cinnamoyl ester hydrolase from the anaerobic fungus Piromyces equi acts synergistically with xylanase and is part of a multiprotein cellulose-binding cellulase-hemicellulase complex

Lookup NU author(s): Professor Harry Gilbert

Downloads

Full text for this publication is not currently held within this repository. Alternative links are provided below where available.


Abstract

A collection of clones, isolated from a Piromyces equi cDNA expression library by immunoscreening with antibodies raised against affinity purified multienzyme fungal cellulase-hemicellulase complex, included one which expressed cinnamoyl ester hydrolase activity. The P. equi cinnamoyl ester hydrolase gene (estA) comprised an open reading frame of 1608 nt encoding a protein (EstA) of 536 amino acids and 55,540 Da. EstA was modular in structure and comprised three distinct domains. The N-terminal domain was closely similar to a highly conserved non-catalytic 40-residue docking domain which is prevalent in cellulases and hemicellulases from three species of anaerobic fungi and binds to a putative scaffolding protein during assembly of the fungal cellulase complex. The second domain was also not required for esterase activity and appeared to be an atypically large linker comprising multiple tandem repeats of a 13-residue motif. The C-terminal 270 residues of EstA contained an esterase catalytic domain that exhibited overall homology with a small family of esterases, including acetylxylan esterase D (XYLD) from Pseudomonas fluorescens subsp. cellulosa and acetylxylan esterase from Aspergillus niger. This region also contained several smaller blocks of residues that displayed homology with domains tentatively identified as containing the essential catalytic residues of a larger group of serine hydrolases. A truncated variant of EstA, comprising the catalytic domain alone (EstA'), was expressed in Escherichia coli as a thioredoxin fusion protein and was purified to homogeneity. EstA' was active against synthetic and plant cell-wall-derived substrates, showed a marked preference for cleaving 1→5 ester linkages between ferulic acid and arabinose in feruloylated arabino-xylo-oligosaccharides and was inhibited by the serine-specific protease inhibitor aminoethylbenzene-sulphonylfluoride. EstA' acted synergistically with xylanase to release more than 60% of the esterified ferulic acid from the arabinoxylan component of plant cell walls. Western analysis confirmed that EstA is produced by P. equi and is a component of the aggregated multienzyme cellulase-hemicellulase complex. Hybrid proteins, harbouring one, two or three iterations of the conserved 40-residue fungal docking domain fused to the reporter protein glutathione S-transferase, were produced. Western blot analysis of immobilized P. equi cellulase-hemicellulase complex demonstrated that each of the hybrid proteins bound to a 97 kDa polypeptide in the extracellular complex.


Publication metadata

Author(s): Fillingham IJ, Kroon PA, Williamson G, Gilbert HJ, Hazlewood GP

Publication type: Article

Publication status: Published

Journal: Biochemical Journal

Year: 1999

Volume: 343

Issue: 1

Pages: 215-224

Print publication date: 27/07/1999

ISSN (print): 0264-6021

ISSN (electronic): 1470-8728

Publisher: Portland Press Ltd.

URL: http://dx.doi.org/10.1042/0264-6021:3430215

DOI: 10.1042/0264-6021:3430215

PubMed id: 10493932


Altmetrics

Altmetrics provided by Altmetric


Actions

Find at Newcastle University icon    Link to this publication


Share