Biochemical Analysis of a β- d -Xylosidase and a Bifunctional Xylanase-Ferulic Acid Esterase from a Xylanolytic Gene Cluster in Prevotella ruminicola 23

Abstract

Prevotella ruminicola 23 is an obligate anaerobic bacterium in the phylum Bacteroidetes that contributes to hemicellulose utilization within the bovine rumen. To gain insight into the cellular machinery that this organism elaborates to degrade the hemicellulosic polymer xylan, we identified and cloned a gene predicted to encode a bifunctional xylanase-ferulic acid esterase (xyn10D-fae1A) and expressed the recombinant protein in Escherichia coli. Biochemical analysis of purified Xyn10D-Fae1A revealed that this protein possesses both endo-␤-1,4-xylanase and ferulic acid esterase activities. A putative glycoside hydrolase (GH) family 3 ␤-Dglucosidase gene, with a novel PA14-like insertion sequence, was identified two genes downstream of xyn10D-fae1A. Biochemical analyses of the purified recombinant protein revealed that the putative ␤-D-glucosidase has activity for pNP-␤-D-xylopyranoside, pNP-␣-L-arabinofuranoside, and xylo-oligosaccharides; thus, the gene was designated xyl3A. When incubated in combination with Xyn10D-Fae1A, Xyl3A improved the release of xylose monomers from a hemicellulosic xylan substrate, suggesting that these two enzymes function synergistically to depolymerize xylan. Directed mutagenesis studies of Xyn10D-Fae1A mapped the catalytic sites for the two enzymatic functionalities to distinct regions within the polypeptide sequence. When a mutation was introduced into the putative catalytic site for the xylanase domain (E280S), the ferulic acid esterase activity increased threefold, which suggests that the two catalytic domains for Xyn10D-Fae1A are functionally coupled. Directed mutagenesis of conserved residues for Xyl3A resulted in attenuation of activity, which supports the assignment of Xyl3A as a GH family 3 ␤-D-xylosidase.␤-1,4-linked xylopyranose is the principal component of plant cell wall hemicellulose, which represents the second largest reservoir of fixed carbon in the biosphere (11,30,34,38,42,72). The catabolic breakdown of hemicellulose thus represents a critical step in the recycling of carbon in nature and has been targeted as a subject of intense research with respect to renewable energy resources. Two enzymes of principal importance for recycling hemicellulosic material are endo-1,4-␤-xylanases (EC 3.2.1.8), which cleave the xylan backbone, and ␤-D-xylosidases (EC 3.2.1.37), which cleave xylose monomers from the nonreducing end of xylo-oligosaccharides (17).Prevotella ruminicola 23 is an important member of the anaerobic rumen microbiota (60) that contributes to the utilization of noncellulosic polysaccharides, such as starch and xylan (15,25,35,55). Despite its documented importance in rumen physiology, relatively little is known about the cellular machinery that P. ruminicola 23 employs to harvest energy from hemicellulosic substrates. Several studies have explored the carbohydrate active enzymes from the related taxon Prevotella bryantii B 1 4 (previously classified as P. ruminicola B 1 4) (25,27,28,50,52,66,73); however, less is known about the xylanolytic system that P. rumi...

Functional Diversity of Four Glycoside Hydrolase Family 3 Enzymes from the Rumen Bacterium Prevotella bryantii B 1 4

“…The pentose sugar ␤-D-xylopyranose does not possess the additional CH 2 OH substituent from C5 that is present in the hexose sugar ␤-D-glucopyranose; thus, it is possible that amino acid residues positioned at this site could function to discriminate between the hexose and the pentose on the basis of steric interactions (12). Amino acid sequence alignments of Xyl3B with other biochemically characterized GH3 enzymes revealed the presence of a glutamic acid residue (Glu115) that aligned with Asp120 from the barley enzyme (see Fig.…”

“…The genome for P. bryantii B 1 4 contains four GH family 3 (GH3) genes, each of which was annotated as coding for an enzyme with ␤-glucosidase activity. Previous efforts to identify genes that are important for xylan degradation by its relative Prevotella ruminicola 23 revealed a GH family 3 protein that was annotated as a ␤-glucosidase enzyme but actually exhibited ␤-xylosidase activity (12). Thus, the goal of the current study was to express these four GH family 3 genes from P. bryantii B 1 4 and to characterize and compare the biochemical properties of their gene products.…”

Transcriptomic Analyses of Xylan Degradation by Prevotella bryantii and Insights into Energy Acquisition by Xylanolytic Bacteroidetes

“…After 15 h, the concentration of reducing ends was estimated using the para-hydroxybenzoic acid hydrazide assay with glucose as the standard (21). For qualitative identification of the hydrolysis products, the reactions were resolved by TLC as described previously (9). For more quantitative analysis of the products of hydrolysis, the wheat arabinoxylan hydrolysates were analyzed by high performance anion exchange chromatography (HPAEC) as described earlier (4).…”

Thermostable feruloyl esterase for the bioproduction of ferulic acid from triticale bran

“…On the bacterial side, even fewer clones are available. These are limited to Cellvibrio japonicum (DeBoy et al 2008; formerly Pseudomonas fluorescens; Ferreira et al 1993), Burkholderia multivorans (Rashamuse et al 2007), Butyrivibrio fibrisolvens (Dalrymple et al 1996;Dalrymple and Swadling 1997), Pseudoalteromonas halosplanktis (Aurilia et al 2008), Clostridium thermocellum cellulosome (Blum et al 2000), and Prevotella ruminicola (Dodd et al 2009). …”