Effect of C-6 Methylol Groups on Substrate Recognition of Glucose/Xylose Mixed Oligosaccharides by Cellobiose Dehydrogenase from the Basidiomycete <i>Phanerochaete chrysosporium</i>

Abstract

Cellobiose dehydrogenase (CDH) is a flavocytochrome catalyzing oxidation of the reducing end of cellobiose and cellooligosaccharides, and has a key role in the degradation of cellulosic biomass by filamentous fungi. Here, we use a lineup of glucose/xylose-mixed β-1,4-linked disaccharides and trisaccharides, enzymatically synthesized by means of the reverse reaction of cellobiose phosphorylase and cellodextrin phosphorylase, to investigate the substrate recognition of CDH. We found that CDH utilizes β-D-xylopyranosyl-(1→4)-D-glucopyranose (Xyl-Glc) as an electron donor with similar K m and k cat values to cellobiose. β-D-Glucopyranosyl-(1→4)-D-xylopyranose (Glc-Xyl) shows a higher K m value, while xylobiose does not serve as a substrate. Trisaccharides show similar behavior; i.e., trisaccharides with cellobiose and Xyl-Glc units at the reducing end show similar kinetics, while the enzyme was less active towards those with Glc-Xyl, and inactive towards those with xylobiose. We also use docking simulation to evaluate substrate recognition of the disaccharides, and we discuss possible molecular mechanisms of substrate recognition by CDH.

Characterization of a novel AA3_1 xylooligosaccharide dehydrogenase from Thermothelomyces myriococcoides CBS 398.93

“…AA3_1 enzymes clearly vary in their ability to oxidize xylooligosaccharides. For instance, the Pc CDH from Basidiomycota was found to primarily accept cellobiose, but could not oxidize xylobiose, xylotriose, or Glc-Glc-Xyl, Xyl-Glc-Xyl, and Glc-Xyl-Xyl [ 34 ], while xylobiose was oxidized by Nc CDHs and Hi CDHs from Ascomycota, the catalytic efficiency was 50 to 2500 times lower than that towards cellobiose [ 10 , 11 ]. By contrast, the affinity and the catalytic efficiency for Tm XdhA towards xylobiose, xylotriose and xylotetraose was more than ten times higher compared to all previous characterized CDHs [ 12 ].…”

Characterization of a novel AA3_1 xylooligosaccharide dehydrogenase from Thermothelomyces myriococcoides CBS 398.93

“…AA3_1 enzymes clearly vary in their ability to oxidize xylooligosaccharides. For instance, the PcCDH from Basidomycota was found to primarily accept cellobiose but could not oxidize xylobiose, xylotriose, or Glc-Glc-Xyl, Xyl-Glc-Xyl, and Glc-Xyl-Xyl [34], while xylobiose was oxidized by NcCDHs and HiCDHs from Ascomycota, the catalytic e ciency was 50 to 2500 times lower than that towards cellobiose [10,11]. By constrast, the a nity and the catalytic e ciency for TmXdhA towards xylobiose, xylotriose and xylotetraose was more than ten-times higher compared to all previous characterized CDHs [12].…”