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

Abstract

Prevotella bryantii B 1 4 is a member of the phylum Bacteroidetes and contributes to the degradation of hemicellulose in the rumen. The genome of P. bryantii harbors four genes predicted to encode glycoside hydrolase (GH) family 3 (GH3) enzymes. To evaluate whether these genes encode enzymes with redundant biological functions, each gene was cloned and expressed in Escherichia coli. Biochemical analysis of the recombinant proteins revealed that the enzymes exhibit different substrate specificities. One gene encoded a cellodextrinase (CdxA), and three genes encoded ␤-xylosidase enzymes (Xyl3A, Xyl3B, and Xyl3C) with different specificities for either para-nitrophenyl (pNP)-linked substrates or substituted xylooligosaccharides. To identify the amino acid residues that contribute to catalysis and substrate specificity within this family of enzymes, the roles of conserved residues (R177, K214, H215, M251, and D286) in Xyl3B were probed by site-directed mutagenesis. Each mutation led to a severely decreased catalytic efficiency without a change in the overall structure of the mutant enzymes. Through amino acid sequence alignments, an amino acid residue (E115) that, when mutated to aspartic acid, resulted in a 14-fold decrease in the k cat /K m for pNP-␤-Dxylopyranoside (pNPX) with a concurrent 1.1-fold increase in the k cat /K m for pNP-␤-D-glucopyranoside (pNPG) was identified. Amino acid residue E115 may therefore contribute to the discrimination between ␤-xylosides and ␤-glucosides. Our results demonstrate that each of the four GH3 enzymes has evolved to perform a specific role in lignopolysaccharide hydrolysis and provide insight into the role of active-site residues in catalysis and substrate specificity for GH3 enzymes.Among the bacterial genera in the bovine rumen, Prevotella species are the most numerically abundant species by both culture counts and by analysis of 16S rRNA abundances (13,43). These organisms contribute to the breakdown of plant protein and hemicellulose within the rumen microbial ecosystem. Despite these important physiological roles, relatively little is known about the molecular mechanisms for plant polysaccharide utilization by ruminal Prevotella species. The genomes for Prevotella bryantii B 1 4 and P. ruminicola 23 have recently been sequenced and harbor a large number of putative glycoside hydrolase (GH) genes

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

“…Transcriptional Analysis of P. bryantii B 1 4 Using Microarrays and RNA-Seq-To assess the reproducibility of the microarray and RNA-Seq analyses, the expression levels for all annotated genes in the partial genome sequence of P. bryantii B 1 4 were compared for the two biological replicates using both technologies. These analyses revealed a high correlation in the expression level for each gene in the two biological replicates derived from either DNA microarray or RNA-Seq technologies (supplemental Fig.…”

“…All the methods were as described in our earlier report (4). Furthermore, the expression and purification of the mutant recombinant proteins were performed as described above for the wild-type (WT) PbXyn5A, BeXyn5A, and BiXyn5A.…”

“…Growth of P. bryantii B 1 4 and Isolation of RNA-P. bryantii B 1 4 was grown anaerobically in a modified chemically defined medium (4) with either wheat arabinoxylan (0.15% w/v) or a mixture containing both xylose (0.0885% w/v) and arabinose (0.0615% w/v) as the sole carbohydrate sources. The concentrations of XA in the mixture were equivalent to that in the WAX polysaccharide (59:41, xylose/arabinose).…”

“…The five genes were cloned into pET-46b by ligation-independent cloning as described previously (4). The recombinant hexahistidine proteins were expressed in E. coli BL21-CodonPlus (DE3) RIL cells, and different purification schemes were used for each protein.…”

“…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).…”