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. 2009 May;191(10):3328-38.
doi: 10.1128/JB.01628-08. Epub 2009 Mar 20.

Biochemical analysis of a beta-D-xylosidase and a bifunctional xylanase-ferulic acid esterase from a xylanolytic gene cluster in Prevotella ruminicola 23

Dylan Dodd  1 , Svetlana A Kocherginskaya, M Ashley Spies, Kyle E Beery, Charles A Abbas, Roderick I Mackie, Isaac K O Cann
Affiliations

Biochemical analysis of a beta-D-xylosidase and a bifunctional xylanase-ferulic acid esterase from a xylanolytic gene cluster in Prevotella ruminicola 23

Dylan Dodd et al. J Bacteriol. 2009 May.

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-beta-1,4-xylanase and ferulic acid esterase activities. A putative glycoside hydrolase (GH) family 3 beta-D-glucosidase 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 beta-D-glucosidase has activity for pNP-beta-D-xylopyranoside, pNP-alpha-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 beta-D-xylosidase.

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Figures

FIG. 1.
FIG. 1.
Identification of a putative xylanase-esterase gene (xyn10D-fae1A) from the genome of P. ruminicola 23. The genome of P. ruminicola 23 was sequenced by the North American Consortium for Fibrolytic Rumen Bacteria in collaboration with The Institute for Genomic Research. ORF02827 was annotated as a putative bifunctional xylanase-esterase. Downstream of the xylanase-esterase are genes predicted to encode a hypothetical protein, a β-d-glucosidase, an ABC transporter, and a hybrid two-component system regulator.
FIG. 2.
FIG. 2.
xyn10D-fae1A encodes a bifunctional xylanase-esterase. (A) Purification of recombinant Xyn10D-Fae1A. The eluate from cobalt chelate chromatography was analyzed by 12% SDS-PAGE, followed by Coomassie brilliant blue G-250 staining. MW, molecular weight (in thousands). (B) Gel filtration chromatography. The size of purified Xyn10D-Fae1A was estimated by size exclusion chromatography. The molecular weight of Xyn10D-Fae1A was calculated from the retention time of the peak absorbance by comparison with calibration standards having known molecular weights. Molecular weight is reported as the mean ± standard deviation from three independent experiments. mAU, milli-absorbance units. (C) Depolymerization of OSX. Xyn10D-Fae1A was assessed for its capacity to depolymerize OSX by incubating the protein on an agar plate infused with OSX followed by staining with Congo red. (D) Hydrolysis of 1-napthyl butyrate. Xyn10D-Fae1A was assessed for its capacity to hydrolyze 1-naphtyl butyrate by incubating the protein on an agar plate infused with 1-NB followed by development with fast garnet GBC sulfate. (E) Hydrolysis of xylo-oligosaccharides. Xyn10D-Fae1A-catalyzed hydrolysis of xylo-oligosaccharides (X2 to X6) was assessed by incubating the enzyme with each substrate and then resolving the products by TLC followed by staining with methanolic orcinol.
FIG. 3.
FIG. 3.
xyl3A encodes a functional β-xylosidase. (A) Purification of recombinant Xyl3A. The eluate from cobalt chelate chromatography was analyzed by 12% SDS-PAGE, followed by Coomassie brilliant blue G-250 staining. MW, molecular weight (in thousands). (B) Gel filtration chromatography. The size of purified Xyl3A was estimated by size exclusion chromatography. The molecular weight (MW, in thousands; reported as the mean ± standard deviation from three independent experiments) of Xyl3A was calculated from the retention time of the peak absorbance by comparison with calibration standards having known molecular weights. mAU, milli-absorbance units. (C) Hydrolysis of pNP-linked sugars. Xyl3A was assessed for its capacity to hydrolyze several pNP-linked sugars by UV spectroscopy. Values are means ± standard deviations from three independent experiments. (D) Hydrolysis of xylo-oligosaccharides. Xyl3A-catalyzed hydrolysis of xylo-oligosaccharides (X2 to X6) was assessed by incubating the enzyme with each substrate and then resolving the products by TLC followed by staining with methanolic orcinol.
FIG. 4.
FIG. 4.
Xyn10D-Fae1A and Xyl3A function synergistically to release xylose from xylan. (A) TLC separation of products released from OSX by Xyn10D-Fae1A. Xyn10D-Fae1A (0.50 μM) or Xyl3A (0.50 μM) was incubated with OSX (1%), and the products were resolved by TLC followed by staining with methanolic orcinol. Xylo-oligosaccharide standards X1 to X4 were spotted onto the plate to serve as markers (lane 1) for the identification of hydrolysis products. The indicated enzymes were incubated with OSX at 37°C for 14 h, and 2.5 μl (lanes 2, 4, 6, and 8) or 5.0 μl (lanes 3, 5, 7, and 9) of the reaction mixtures was spotted onto the TLC plate. (B) Reducing sugars released from OSX by Xyn10D-Fae1A and Xyl3A. Wild-type or mutant (E280S or S629A) Xyn10D-Fae1A was incubated with OSX (1%), and the reducing sugars were detected by using the PAHBAH assay. The reducing sugar concentrations were calculated from the absorbance at 410 nm with comparison to a standard curve generated with known concentrations of glucose. Two-tailed P values were determined by performing an unpaired Student's t test.
FIG. 5.
FIG. 5.
Mutational analyses of Xyn10D-Fae1A map the two catalytic domains to discrete regions of the polypeptide sequence. (A) Domain architecture for Xyn10D-Fae1A. Functional domains were assigned utilizing the Pfam online server (24). Amino acid substitutions were independently introduced into the putative catalytic residue for the xylanase domain (Glu280) and the ferulic acid esterase domain (Ser629) by site-directed mutagenesis. (B) Depolymerization of OSX by wild-type and mutant (E280S or S629A) Xyn10D-Fae1A. Wild-type or mutant (E280S or S629A) Xyn10D-Fae1A was incubated with OSX (1%), and the products were resolved by TLC followed by staining with methanolic orcinol. Xylo-oligosaccharide standards X1 to X4 were spotted on the plate in lane 1 to serve as markers for the identification of hydrolysis products. The wild-type enzyme, E280S mutant, or S629A mutant (0.93 μM in lanes 3, 5, and 7, respectively, and 1.9 μM in lanes 4, 6, and 8, respectively) was incubated with OSX at 37°C for 21 h, and 2.5 μl of the reaction mixtures was spotted onto the TLC plate. (C) Reducing sugars released from OSX by wild-type and mutant (E280S or S629A) Xyn10D-Fae1A. Wild-type or mutant (E280S or S629A) Xyn10D-Fae1A was incubated with OSX (1%), and the reducing sugars were detected by using the PAHBAH assay. The reducing sugar concentrations were calculated from the absorbance at 410 nm with comparison to a standard curve generated with known concentrations of glucose. (D) Hydrolysis of methyl ferulate by wild-type and mutant (E280S or S629A) Xyn10D-Fae1A. Methyl ferulate (5 mM) was incubated with wild-type, E280S, or S629A Xyn10D-Fae1A (1.2 μM, final concentration). Following incubation at 37°C for 30 min, ferulic acid was determined by HPLC as described by Wang et al. (67). For panels C and D, values are means plus standard deviations from three independent experiments. Two-tailed P values were determined by performing an unpaired Student's t test.
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References

    1. Aurilia, V., J. C. Martin, S. I. McCrae, K. P. Scott, M. T. Rincon, and H. J. Flint. 2000. Three multidomain esterases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 that carry divergent dockerin sequences. Microbiology 1461391-1397. - PubMed
    1. Avgustin, G., H. J. Flint, and T. R. Whitehead. 1992. Distribution of xylanase genes and enzymes among strains of Prevotella (Bacteroides) ruminicola from the rumen. FEMS Microbiol. Lett. 78137-143. - PubMed
    1. Bergman, E. N. 1990. Energy contributions of volatile fatty acids from the gastrointestinal tract in various species. Physiol. Rev. 70567-590. - PubMed
    1. Bertani, G. 2004. Lysogeny at mid-twentieth century: P1, P2, and other experimental systems. J. Bacteriol. 186595-600. - PMC - PubMed
    1. Bertani, G. 1951. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacteriol. 62293-300. - PMC - PubMed

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