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

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.

SUBMITTER: Igarashi K

PROVIDER: S-EPMC8293687 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundCellobiose dehydrogenase from Phanerochaete chrysosporium (PcCDH) is a key enzyme in lignocellulose depolymerization, biosensors and biofuel cells. For these applications, it should retain important molecular and catalytic properties when recombinantly expressed. While homologous expression is time-consuming and the prokaryote Escherichia coli is not suitable for expression of the two-domain flavocytochrome, the yeast Pichia pastoris is hyperglycosylating the enzyme. Fungal expression hosts like Aspergillus niger and Trichoderma reesei were successfully used to express CDH from the ascomycete Corynascus thermophilus. This study describes the expression of basidiomycetes PcCDH in T. reesei (PcCDHTr) and the detailed comparison of its molecular, catalytic and electrochemical properties in comparison with PcCDH expressed by P. chrysosporium and P. pastoris (PcCDHPp).ResultsPcCDHTr was recombinantly produced with a yield of 600 U L-1 after 4 days, which is fast compared to the secretion of the enzyme by P. chrysosporium. PcCDHTr and PcCDH were purified to homogeneity by two chromatographic steps. Both enzymes were comparatively characterized in terms of molecular and catalytic properties. The pH optima for electron acceptors are identical for PcCDHTr and PcCDH. The determined FAD cofactor occupancy of 70% for PcCDHTr is higher than for other recombinantly produced CDHs and its catalytic constants are in good accordance with those of PcCDH. Mass spectrometry showed high mannose-type N-glycans on PcCDH, but only single N-acetyl-D-glucosamine additions at the six potential N-glycosylation sites of PcCDHTr, which indicates the presence of an endo-N-acetyl-β-D-glucosaminidase in the supernatant.ConclusionsHeterologous production of PcCDHTr is faster and the yield higher than secretion by P. chrysosporium. It also does not need a cellulose-based medium that impedes efficient production and purification of CDH by binding to the polysaccharide. The obtained high uniformity of PcCDHTr glycoforms will be very useful to investigate electron transfer characteristics in biosensors and biofuel cells, which are depending on the spatial restrictions inflicted by high-mannose N-glycan trees. The determined catalytic and electrochemical properties of PcCDHTr are very similar to those of PcCDH and the FAD cofactor occupancy is good, which advocates T. reesei as expression host for engineered PcCDH for biosensors and biofuel cells.

Dataset Information

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

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