Journal article
Biochemical Analysis of a β-d-Xylosidase and a Bifunctional Xylanase-Ferulic Acid Esterase from a Xylanolytic Gene Cluster in Prevotella ruminicola 23
Journal of bacteriology, Vol.191(10), pp.3328-3338
03/20/2009
DOI: 10.1128/JB.01628-08
PMCID: PMC2687155
PMID: 19304844
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-β-1,4-xylanase and ferulic acid esterase activities. A putative glycoside hydrolase (GH) family 3 β-
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 β-
d
-glucosidase has activity for
p
NP-β-
d
-xylopyranoside,
p
NP-α-
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 β-
d
-xylosidase.
Details
- Title: Subtitle
- Biochemical Analysis of a β-d-Xylosidase and a Bifunctional Xylanase-Ferulic Acid Esterase from a Xylanolytic Gene Cluster in Prevotella ruminicola 23
- Creators
- Dylan Dodd - University of Illinois Urbana-ChampaignSvetlana A. Kocherginskaya - Department of Animal SciencesM. Ashley Spies - University of Illinois Urbana-ChampaignKyle E. Beery - Archer Daniels MidlandCharles A. Abbas - Archer Daniels MidlandRoderick I. Mackie - University of Illinois Urbana-ChampaignIsaac K. O. Cann - University of Illinois Urbana-Champaign
- Resource Type
- Journal article
- Publication Details
- Journal of bacteriology, Vol.191(10), pp.3328-3338
- Publisher
- American Society for Microbiology (ASM)
- DOI
- 10.1128/JB.01628-08
- PMID
- 19304844
- PMCID
- PMC2687155
- ISSN
- 0021-9193
- eISSN
- 1098-5530
- Language
- English
- Date published
- 03/20/2009
- Academic Unit
- Pharmaceutical Sciences and Experimental Therapeutics; Biochemistry and Molecular Biology; Medicinal and Natural Products Chemistry
- Record Identifier
- 9984293087302771
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