A novel member of glycoside hydrolase family 30 subfamily 8 with altered substrate specificity.
Endoxylanases classified into glycoside hydrolase family 30 subfamily 8 (GH30-8) are known to hydrolyze the hemicellulosic polysaccharide glucuronoxylan (GX) but not arabinoxylan or neutral xylooligosaccharides. This is owing to the specificity of these enzymes for the α-1,2-linked glucuronate (GA) appendage of GX. Limit hydrolysis of this substrate produces a series of aldouronates each containing a single GA substituted on the xylose penultimate to the reducing terminus. In this work, the structural and biochemical characterization of xylanase 30A from Clostridium papyrosolvens (CpXyn30A) is presented. This xylanase possesses a high degree of amino-acid identity to the canonical GH30-8 enzymes, but lacks the hallmark β8-α8 loop region which in part defines the function of this GH30 subfamily and its role in GA recognition. CpXyn30A is shown to have a similarly low activity on all xylan substrates, while hydrolysis of xylohexaose revealed a competing transglycosylation reaction. These findings are directly compared with the model GH30-8 enzyme from Bacillus subtilis, XynC. Despite its high sequence identity to the GH30-8 enzymes, CpXyn30A does not have any apparent specificity for the GA appendage. These findings confirm that the typically conserved β8-α8 loop region of these enzymes influences xylan substrate specificity but not necessarily β-1,4-xylanase function.
Forest Products Laboratory, USDA Forest Service, Madison, Wisconsin, USA.
Franz J
FJ
St John
Forest Products Laboratory, USDA Forest Service, Madison, Wisconsin, USA.
Diane
D
Dietrich
Forest Products Laboratory, USDA Forest Service, Madison, Wisconsin, USA.
Casey
C
Crooks
Department of Biochemistry and Molecular Biology, University of Maryland, Maryland, USA.
Edwin
E
Pozharski
Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico, USA.
Javier M
JM
González
Department of Chemistry, Physics and Geology, Winthrop University, Rock Hill, South Carolina, USA.
Elizabeth
E
Bales
Department of Chemistry, Physics and Geology, Winthrop University, Rock Hill, South Carolina, USA.
Kennon
K
Smith
Department of Chemistry, Physics and Geology, Winthrop University, Rock Hill, South Carolina, USA.
Jason C
JC
Hurlbert
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology; D003013:Clostridium / Q000737:chemistry / Q000201:enzymology*; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016415:Sequence Alignment; D013379:Substrate Specificity; D014995:Xylosidases / Q000737:chemistry* / Q000378:metabolism
D014995:Xylosidases
glycoside hydrolase family 30; low-value biomass; wildfire; xylanase