title: A single amino acid change (Y318F) in the L-arabitol dehydrogenase (LadA) from Aspergillus niger results in a significant increase in affinity for D-sorbitol.
abstract: BACKGROUND
  L-arabitol dehydrogenase (LAD) and xylitol dehydrogenase (XDH) are involved in the degradation of L-arabinose and D-xylose, which are among the most abundant monosaccharides on earth. Previous data demonstrated that LAD and XDH not only differ in the activity on their biological substrate, but also that only XDH has significant activity on D-sorbitol and may therefore be more closely related to D-sorbitol dehydrogenases (SDH). In this study we aimed to identify residues involved in the difference in substrate specificity.
  
  
  RESULTS
  Phylogenetic analysis demonstrated that LAD, XDH and SDH form 3 distinct groups of the family of dehydrogenases containing an Alcohol dehydrogenase GroES-like domain (pfam08240) and likely have evolved from a common ancestor. Modelling of LadA and XdhA of the saprobic fungus Aspergillus niger on human SDH identified two residues in LadA (M70 and Y318), that may explain the absence of activity on D-sorbitol. While introduction of the mutation M70F in LadA of A. niger resulted in a nearly complete enzyme inactivation, the Y318F resulted in increased activity for L-arabitol and xylitol. Moreover, the affinity for D-sorbitol was increased in this mutant.
  
  
  CONCLUSIONS
  These data demonstrates that Y318 of LadA contributes significantly to the substrate specificity difference between LAD and XDH/SDH.
authors.affiliation: Department of Crystal and Structural Chemistry, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands. lucy.rutten@gmail.com
authors.forename: Lucy
authors.initials: L
authors.lastname: Rutten
authors.forename: Cecile
authors.initials: C
authors.lastname: Ribot
authors.forename: Blanca
authors.initials: B
authors.lastname: Trejo-Aguilar
authors.forename: Han A B
authors.initials: HA
authors.lastname: Wösten
authors.forename: Ronald P
authors.initials: RP
authors.lastname: de Vries
mesh_terms: D019943:Amino Acid Substitution; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D050542:D-Xylulose Reductase / Q000235:genetics / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics; D005800:Genes, Fungal; D007064:L-Iditol 2-Dehydrogenase / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D013012:Sorbitol / Q000378:metabolism*; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics / Q000378:metabolism*
chemical_list: D004271:DNA, Fungal; D013012:Sorbitol; D013401:Sugar Alcohol Dehydrogenases; D007064:L-Iditol 2-Dehydrogenase; D050542:D-Xylulose Reductase
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