Xylose reductase (XR) from Candida tenuis (CtXR) is a structurally characterized member of family 2 of the aldo-keto reductase (AKR) superfamily of proteins, its family designation being AKR2B5. The enzyme is composed of two identical subunits that contact each other in a largely hydrated, predominantly polar interface. An important question not clearly answered by CtXR structure pertains to the relationship of oligomerization and enzyme activity. In an effort to destabilize the wild-type dimer, the most important secondary structural element of the CtXR interface, the alpha5 helix, was altered by site-directed mutagenesis. Ala-173 and Leu-174 were replaced individually by arginine, and Arg-180 was changed into alanine. A173R and L174R mutants did not fold properly during recombinant protein production in Escherichia coli and could not be isolated. Like the wild type, the R180A mutant is a dimer in solution which does not dissociate into subunits under mild urea conditions (</=2 M). Catalytic efficiency (k(cat)/K(xylose)) and turnover number (k(cat)) of the R180A mutant for NADH-dependent reduction of D-xylose are both approximately 2.5-fold decreased compared to corresponding kinetic parameters of the wild type. Differences in kinetic isotope effects for the mutant (Dk(cat)=1.0; Dk(cat)/K(xylose)=1.9) and the wild type (Dk(cat)=1.5; Dk(cat)/K(xylose)=2.8) suggest subtle changes in catalytic function as result of the mutation. Therefore, altering interactions at the dimer interface may have long range effects that were not predictable from the X-ray structure.