The amino acid sequence of ribitol dehydrogenase-F, a mutant enzyme with improved xylitol dehydrogenase activity

A mutant ribitol dehydrogenase (RDH-F) was purified from Klebsiella aerogen es strain F which evolved from the wild-type strain A under selective press ure to improve growth on xylitol, a poor substrate used as sole carbon sour ce. The ratio of activities on xylitol (500 mM) and ribitol (50 mM) was 0.1 54 for RDH-F compared to 0.033 for the wild-type (RDH-A) enzyme. The comple te amino acid sequence of RDH-F showed the mutations. Q60 for E60 and V215 for L215 in the single polypeptide chain of 249 amino acid residues. Struct ural modeling based on homologies with two other microbial dehydrogenases s uggests that E60 --> Q60 is a neutral mutation, since it lies in a region f ar from the catalytic site and should not cause structural perturbations. I n contrast, L215 --> V215 lies in variable region II and would shift a loop that interacts with the NADH cofactor. Another improved ribitol dehydrogen ase, RDH-D, contains an A196 --> P196 mutation that would disrupt a surface alpha-helix in region II. Hence conformational changes in this region appe ar to be responsible for the improved xylitol specificity.