Three multidomain esterases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 that carry divergent dockerin sequences The GenBank accession numbers for the sequences reported in this paper are AJ238716 (cesA) and AJ272430 (xynE).

Abstract

Three enzymes carrying esterase domains have been identified in the rumen cellulolytic anaerobe Ruminococcus flavefaciens 17. The newly characterized CesA gene product (768 amino acids) includes an N-terminal acetylesterase domain and an unidentified C-terminal domain, while the previously characterized XynB enzyme (781 amino acids) includes an internal acetylesterase domain in addition to its N-terminal xylanase catalytic domain. A third gene, xynE, is predicted to encode a multidomain enzyme of 792 amino acids including a family 11 xylanase domain and a C-terminal esterase domain. The esterase domains from CesA and XynB share significant sequence identity (44 %) and belong to carbohydrate esterase family 3 ; both domains are shown here to be capable of deacetylating acetylated xylans, but no evidence was found for ferulic acid esterase activity. The esterase domain of XynE, however, shares 42 % amino acid identity with a family 1 phenolic acid esterase domain identified from Clostridum thermocellum XynZ. XynB, XynE and CesA all contain dockerin-like regions in addition to their catalytic domains, suggesting that these enzymes form part of a cellulosome-like multienzyme complex. The dockerin sequences of CesA and XynE differ significantly from those previously described in R. flavefaciens polysaccharidases, including XynB, suggesting that they might represent distinct dockerin specificities.

Unconventional Mode of Attachment of the Ruminococcus flavefaciens Cellulosome to the Cell Surface

“…flavefaciens strain 17 produces a cellulosome complex that is known to involve the cohesin-containing structural components ScaA, ScaB, and ScaC, together with interacting enzymes and unidentified proteins that carry dockerin domains (1,(36)(37)(38). The assembly of the different components in the R. flavefaciens cellulosome differs from the proposed molecular architecture in the clostridial cellulosomes.…”

Novel Organization and Divergent Dockerin Specificities in the Cellulosome System of Ruminococcus flavefaciens

“…Owing to the distinctive variation in dockerin sequences between different enzymes from R. flavefaciens 17 (1), it was of interest to assess the ability of other dockerin-containing enzyme subunits from R. flavefaciens to bind to ScaA. Appropriate catalytically active fragments from the enzymes EndB, XynB, XynE, and CesA (1,15,38) were all overexpressed and purified as His-tagged products after cloning in pET28a vectors. The interaction of the resultant dockerin-containing fragments to the purified ScaA-Coh2 domain was examined in a series of Western blotting experiments, using biotinylated ScaA-Coh2 as the probe in each case.…”

“…The R. flavefaciens cohesins were considered to constitute a new group, designated type III (11). Furthermore the dockerins identified in R. flavefaciens enzymes also show among themselves a high degree of sequence divergence (1). In view of these findings, the present investigation explores further the organization of plant cell wall-degrading enzyme complexes in R. flavefaciens.…”

Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases

“…Some AcXEs are produced as bimodular or multimodular enzymes [37,38]. Frequently their catalytic domain is linked to a different catalytic module or to a carbohydrate binding module (CBM), emphasizing their role in plant cell wall degradation.…”