Functional diversity of four glycoside hydrolase family 3 enzymes from the rumen bacterium Prevotella bryantii B14

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 beta-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-beta-d-xylopyranoside (pNPX) with a concurrent 1.1-fold increase in the k(cat)/K(m) for pNP-beta-d-glucopyranoside (pNPG) was identified. Amino acid residue E115 may therefore contribute to the discrimination between beta-xylosides and beta-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.

FIG. 1.

GH family 3 genes from P. bryantii B₁4 encode functional enzymes. (A) Purification of recombinant GH3 proteins. The eluate from nickel-chelate chromatography was analyzed by 12% SDS-PAGE, followed by Coomassie brilliant blue G-250 staining. MW, molecular weight (in thousands). (B to E) Hydrolysis of pNP-linked sugars. CdxA (B), Xyl3A (C), Xyl3B (D), and Xyl3C (E) were assessed for their capacities to hydrolyze several pNP-linked sugars by UV spectroscopy. pNPX, pNPA, pNPG, and pNPG₂ represent pNP-β-d-xylopyranoside, pNP-α-l-arabinopyranoside, pNP-β-d-glucopyranoside, and pNP-β-d-cellobioside, respectively.

FIG. 2.

Hydrolysis of cellohexaose and xylohexaose by P. bryantii B₁4 GH family 3 enzymes. The hydrolysis of cellohexaose and xylohexaose was assessed by incubating the enzyme with each substrate and then resolving the products by high-performance anion-exchange chromatography (HPAEC) followed by detection with a pulsed amperometric detector (PAD). The hydrolysis products were identified by comparisons of peaks with retention times of purified substrates. Abbreviations for oligosaccharides are as follows: glucose through cellohexaose, G₁ to G₆; xylose through xylohexaose, X₁ to X₆.

FIG. 3.

Xyl3B and Xyl3C exhibit differences in specificity for 4-O-methyl glucuronic acid-substituted xylooligosaccharides. Shown are data for the hydrolysis of aldouronic acids. P. bryantii B₁4 Xyl3B (A) or Xyl3C (B) was incubated with aldouronic acids in the presence or absence of a GH family 67 α-glucuronidase enzyme, which was cloned from Prevotella bryantii B₁4 (Agu67A), and the hydrolytic products were analyzed by HPAEC-PAD. The hydrolysis products were identified by comparisons of peaks with retention times of purified substrates.

FIG. 4.

Expression of GH3 genes during growth of P. bryantii B₁4 with wheat arabinoxylan and glucose. (A) P. bryantii B₁4 was cultured in a chemically defined growth medium with either wheat arabinoxylan (0.15%, wt/vol) or glucose (0.18%, wt/vol) as the sole carbohydrate source, and the OD₆₀₀ values over time were monitored. (B) At an optical density of 0.2 (mid-log phase of growth), cells were harvested, RNA was extracted, and quantitative reverse transcription-PCR experiments were performed as described in Materials and Methods. Four technical replicates of the Q-PCR were performed for each of three independent biological replicates, and data are reported as means ± standard errors from the mean.

FIG. 5.

GH family 3 active-site residues. (A) Active-site residues for the β-glucan exohydrolase (ExoI) from barley (Hordeum vulgare) bound to glucose are shown (PDB accession no. 1EX1). The residues in parentheses are the corresponding residues in Xyl3B that align with the barley ExoI residues. (B) Predicted model for discrimination between xylosides and glucosides by GH3 enzymes. Asp120 from barley ExoI forms hydrogen bond contacts with the 4′OH and 6′OH groups of glucose. Glu115 from P. bryantii B₁4 Xyl3B is predicted to form hydrogen bond contacts with the 4′OH of xylose and may discriminate between glucose and xylose on the basis of steric interactions.