Relief of Xylose Binding to Cellobiose Phosphorylase by a Single Distal Mutation.
Cellobiose phosphorylase (CBP) cleaves cellobiose-abundant in plant biomass-to glucose and glucose 1-phosphate. However, the pentose sugar xylose, also abundant in plant biomass, acts as a mixed-inhibitor and a substrate for the reverse reaction, limiting the industrial potential of CBP. Preventing xylose, which lacks only a single hydroxymethyl group relative to glucose, from binding to the CBP active site poses a spatial challenge for protein engineering, since simple steric occlusion cannot be used to block xylose binding without also preventing glucose binding. Using CRISPR-based chromosomal library selection, we identified a distal mutation in CBP, Y47H, responsible for improved cellobiose consumption in the presence of xylose. In silico analysis suggests this mutation may alter the conformation of the cellobiose phosphorylase dimer complex to reduce xylose binding to the active site. These results may aid in engineering carbohydrate phosphorylases for improved specificity in biofuel production, and also in the production of industrially important oligosaccharides.
Department of Plant and Microbial Biology, University of California , Berkeley, California 94720, United States.
Kulika
K
Chomvong
Department of Chemical and Biomolecular Engineering, University of California , Berkeley, California 94720, United States.
Eric
E
Lin
Department of Chemistry, University of California , Berkeley, California 94720, United States.
Michael
M
Blaisse
Department of Molecular and Cell Biology, University of California , Berkeley, California 94720, United States.
Abigail E
AE
Gillespie
Department of Chemistry, University of California , Berkeley, California 94720, United States.|Department of Molecular and Cell Biology, University of California , Berkeley, California 94720, United States.|Physical Biosciences Division, Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.
Jamie H D
JH
Cate
D056804:Biofuels; D018533:Biomass; D020134:Catalytic Domain / Q000235:genetics; D002475:Cellobiose / Q000378:metabolism; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D005947:Glucose / Q000378:metabolism; D005964:Glucosyltransferases / Q000378:metabolism*; D009154:Mutation / Q000235:genetics*; D009844:Oligosaccharides / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D014994:Xylose / Q000235:genetics* / Q000378:metabolism*; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
D056804:Biofuels; D009844:Oligosaccharides; D002475:Cellobiose; D014994:Xylose; D005964:Glucosyltransferases; C034714:cellobiose phosphorylase; D005947:Glucose
cellobiose; inhibitor; phosphorylase; protein engineering; xylose