Thermal-induced conformational changes in the product release area drive the enzymatic activity of xylanases 10B: Crystal structure, conformational stability and functional characterization of the xylanase 10B from Thermotoga petrophila RKU-1.
Endo-xylanases play a key role in the depolymerization of xylan and recently, they have attracted much attention owing to their potential applications on biofuels and paper industries. In this work, we have investigated the molecular basis for the action mode of xylanases 10B at high temperatures using biochemical, biophysical and crystallographic methods. The crystal structure of xylanase 10B from hyperthermophilic bacterium Thermotoga petrophila RKU-1 (TpXyl10B) has been solved in the native state and in complex with xylobiose. The complex crystal structure showed a classical binding mode shared among other xylanases, which encompasses the -1 and -2 subsites. Interestingly, TpXyl10B displayed a temperature-dependent action mode producing xylobiose and xylotriose at 20°C, and exclusively xylobiose at 90°C as assessed by capillary zone electrophoresis. Moreover, circular dichroism spectroscopy suggested a coupling effect of temperature-induced structural changes with this particular enzymatic behavior. Molecular dynamics simulations supported the CD analysis suggesting that an open conformational state adopted by the catalytic loop (Trp297-Lys326) provokes significant modifications in the product release area (+1,+2 and +3 subsites), which drives the enzymatic activity to the specific release of xylobiose at high temperatures.
Laboratório Nacional de Biociências (LNBio), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP, Brazil.
Camila Ramos
CR
Santos
Andreia Navarro
AN
Meza
Zaira Bruna
ZB
Hoffmam
Junio Cota
JC
Silva
Thabata Maria
TM
Alvarez
Roberto
R
Ruller
Guilherme Menegon
GM
Giesel
Hugo
H
Verli
Fabio Marcio
FM
Squina
Rolf Alexander
RA
Prade
Mario Tyago
MT
Murakami
D001419:Bacteria / Q000201:enzymology*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004187:Disaccharides / Q000096:biosynthesis; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004795:Enzyme Stability; D006358:Hot Temperature; D011487:Protein Conformation; D017433:Protein Structure, Secondary
D004187:Disaccharides; D043364:Endo-1,4-beta Xylanases; C004173:xylobiose