Characterization of a family 43 β-xylosidase from the xylooligosaccharide utilizing putative probiotic Weissella sp. strain 92.
In this work, we present the first XOS degrading glycoside hydrolase from Weissella, WXyn43, a two-domain enzyme from GH43. The gene was amplified from genomic DNA of the XOS utilizing Weissella strain 92, classified under the species-pair Weissella cibaria/W.confusa, and expressed in Escherichia coli. The enzyme is lacking a putative signal peptide and is, from a homology model, shown to be composed of an N-terminal 5-fold β-propeller catalytic domain and a C-terminal β-sandwich domain of unknown function. WXyn43 hydrolyzed short (1-4)-β-D-xylooligosaccharides, with similar kcat/KM for xylobiose (X2) and xylotriose (X3) and clearly lower efficiency in xylotetraose (X4) conversion. WXyn43 displays the highest reported kcat for conversion of X3 (900 s(-1) at 37 °C) and X4 (770 s(-1)), and kcat for hydrolysis of X2 (907 s(-1)) is comparable with or greater than the highest previously reported. The purified enzyme adopted a homotetrameric state in solution, while a truncated form with isolated N-terminal catalytic domain adopted a mixture of oligomeric states and lacked detectable activity. The homology model shows that residues from both domains are involved in monomer-monomer hydrogen bonds, while the bonds creating dimer-dimer interactions only involved residues from the N-terminal domain. Docking of X2 and X3 in the active site shows interactions corresponding to subsites -1 and +1, while presence of a third subsite is unclear, but interactions between a loop and the reducing-end xylose of X3 may be present.
Biotechnology, Department of Chemistry, Lund University, Lund, Sweden peter.falck@biotek.lu.se.
Peter
P
Falck
Biotechnology, Department of Chemistry, Lund University, Lund, Sweden.
Javier A
JA
Linares-Pastén
Biotechnology, Department of Chemistry, Lund University, Lund, Sweden.
Patrick
P
Adlercreutz
Biotechnology, Department of Chemistry, Lund University, Lund, Sweden.
Eva Nordberg
EN
Karlsson
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D004795:Enzyme Stability; D005965:Glucuronates / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism*; D019936:Probiotics / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity; D058836:Weissella / Q000201:enzymology* / Q000378:metabolism; D014995:Xylosidases / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D005965:Glucuronates; D009844:Oligosaccharides; C570991:xylooligosaccharide; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase
W. cibaria; W. confusa; prebiotics; structure–function; xylosidase