The C-terminal region of xylanase domain in Xyn11A from Paenibacillus curdlanolyticus B-6 plays an important role in structural stability.
Paenibacillus curdlanolyticus B-6 produces an extracellular multienzyme complex containing a major xylanase subunit, designated Xyn11A, which includes two functional domains belonging to glycosyl hydrolase family-11 (GH11) and carbohydrate binding module family-36 (CBM36) and possesses a glycine and asparagine-rich linker (linker). To clarify the roles of each functional domain, recombinant proteins XynXL and XynX (CBM36 deleted and CBM36 and linker deleted, respectively) were constructed. Their xylanase activities were similar toward soluble xylan, whereas XynXL showed decreased hydrolysis activity toward insoluble xylan while XynX had no xylanase activity. To determine the significance of the linker and its neighbor region, XynX was subjected to secondary structural alignments using circular dichroism (CD) spectroscopy and three-dimensional (3D) structural analysis. A seven amino acid (NTITIGG) neighbor linker sequence was highly conserved among GH11 xylanases of Paenibacillus species. Although XynX exhibited a typical GH11 xylanase structure, conformational gaps were observed in the β6- and β12-sheets and in CD spectra. Flipping of the Arg163 side chains in the subsite was also observed upon analysis of superimposed models. Docking analysis using xylohexaose indicated that flipping of the Arg163 side chains markedly affected substrate binding in the subsite. To identify the amino acids related to stabilizing the substrate binding site, XynX with an extended C-terminal region was designed. At least seven amino acids were necessary to recover substrate binding and xylanase activity. These results indicated that the seven amino acid neighbor Xyn11A linker plays an important role in the activity and conformational stability of the xylanase domain.
Department of Chemical Technology, Faculty of Science and Technology, Suan Dusit Rajabhat University, 295 Rajasrima Rd., Dusit, Bangkok, 10300, Thailand.
Junjarus
J
Sermsathanaswadi
Somsak
S
Pianwanit
Patthra
P
Pason
Rattiya
R
Waeonukul
Chakrit
C
Tachaapaikoon
Khanok
K
Ratanakhanokchai
Krisna
K
Septiningrum
Akihiko
A
Kosugi
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D056507:Paenibacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D055550:Protein Stability; D016415:Sequence Alignment; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D014995:Xylosidases