Biochemical analysis of a beta-D-xylosidase and a bifunctional xylanase-ferulic acid esterase from a xylanolytic gene cluster in Prevotella ruminicola 23.
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.
Dodd
Dylan
D
Department of Microbiology, University of Illinois, 1207 W Gregory Drive, Urbana, IL 61801, USA.
Kocherginskaya
Svetlana A
SA
Spies
M Ashley
MA
Beery
Kyle E
KE
Abbas
Charles A
CA
Mackie
Roderick I
RI
Cann
Isaac K O
IK
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002109:Caffeic Acids / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D043364:Endo-1,4-beta Xylanases / Q000235:genetics / Q000378:metabolism; D006027:Glycosides / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D045243:Prevotella ruminicola / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity; D014995:Xylosidases / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D002109:Caffeic Acids; D006027:Glycosides; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase; D043364:Endo-1,4-beta Xylanases; C530806:methyl ferulate