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

A collection of clones, isolated from a Piromyces equi cDNA expression libr ary by immunoscreening with antibodies raised against affinity purified mul tienzyme fungal cellulase-hemicellulase complex, included one which express ed cinnamoyl ester hydrolase activity. The P. equi cinnamoyl ester hydrolas e 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 simila r to a highly conserved non-catalytic 40-residue docking domain which is pr evalent in cellulases and hemicellulases from three species of anaerobic fu ngi and binds to a putative scaffolding protein during assembly of the fung al 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 c ontained an esterase catalytic domain that exhibited overall homology with a small family of esterases, including acetylxylan esterase D (XYLD) from P seudomonas fluorescens subsp. cellulosa and acetylxylan esterase from Asper gillus 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'), w as expressed in Escherichia coli as a thioredoxin fusion protein and was pu rified to homogeneity. EstA' was active against synthetic and plant cell-wa ll-derived substrates, showed a marked preference for cleaving 1 --> 5 este r linkages between ferulic acid and arabinose in feruloylated arabino-xylo- oligosaccharides and was inhibited by the serine-specific protease inhibito r aminoethylbenzene-sulphonylfluoride. EstA' acted synergistically with xyl anase to release more than 60% of the esterified ferulic acid from the arab inoxylan component of plant cell walls. Western analysis confirmed that Est A is produced by P. equi and is a component of the aggregated multienzyme c ellulase-hemicellulase complex. Hybrid proteins, harbouring one, two or thr ee iterations of the conserved 40-residue fungal docking domain fused to th e reporter protein glutathione S-transferase, were produced. Western blot a nalysis of immobilized P. equi cellulase-hemicellulase complex demonstrated that each of the hybrid proteins bound to a 97 kDa polypeptide in the extr acellular complex.