Structure-guided engineering of xylitol dehydrogenase cosubstrate specificity.
Xylitol dehydrogenase (XDH) is one of several enzymes responsible for assimilating xylose into eukaryotic metabolism and is useful for fermentation of xylose contained in agricultural byproducts to produce ethanol. For efficient xylose utilization at high flux rates, cosubstrates should be recycled between the NAD+-specific XDH and the NADPH-preferring xylose reductase, another enzyme in the pathway. To understand and alter the cosubstrate specificity of XDH, we determined the crystal structure of the Gluconobacter oxydans holoenzyme to 1.9 angstroms resolution. The structure reveals that NAD+ specificity is largely conferred by Asp38, which interacts with the hydroxyls of the adenosine ribose. Met39 stacked under the purine ring and was also located near the 2' hydroxyl. Based on the location of these residues and on sequence alignments with related enzymes of various cosubstrate specificities, we constructed a double mutant (D38S/M39R) that was able to exclusively use NADP+, with no loss of activity.
Section of Molecular and Cellular Biology, University of California, Davis, Davis, California 95616, USA.
Andreas H
AH
Ehrensberger
Robert A
RA
Elling
David K
DK
Wilson
D002352:Carrier Proteins / Q000378:metabolism; D020134:Catalytic Domain; D050542:D-Xylulose Reductase / Q000737:chemistry* / Q000235:genetics; D020576:Gluconobacter / Q000201:enzymology*; D020035:Holoenzymes / Q000737:chemistry*; D008274:Magnesium / Q000378:metabolism; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism / Q000493:pharmacokinetics; D028044:Phosphate-Binding Proteins; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014994:Xylose / Q000378:metabolism
C112303:CAVIN2 protein, human; D002352:Carrier Proteins; D020035:Holoenzymes; D008670:Metals; D028044:Phosphate-Binding Proteins; D009243:NAD; D009249:NADP; D014994:Xylose; D050542:D-Xylulose Reductase; D008274:Magnesium