Abstract / Summary
Structure-based mutagenesis was performed to modify the substrate specificity of bacterial l -galactose dehydrogenase from Luteolibacter sp. strain LG18, which also exhibits intrinsic l -glucose dehydrogenase activity. Based on previously reported crystal structures of the enzyme in ternary complex with l -galactose or l -glucose and NADP + , an M212Q mutant was generated and found to exhibit a pronounced preference for l -glucose. Kinetic analysis showed that the M212Q mutant displayed an approximately 20-fold higher k cat / K M value for l -glucose than for l -galactose. To elucidate the structural basis of this altered specificity, the X-ray crystal structure of the M212Q mutant complexed with l -glucose and NADP + was determined. Substitution of Met212 with Gln is assumed to disrupt the hydrophobic environment between the residue and the substrates, which may prevent proper binding of l -galactose and resulting in an approximately 350-fold increase in K M compared with the wild type. In contrast, in the l -glucose–bound M212Q structure, a compensatory hydrogen bond is formed between the equatorial 4-OH group of l -glucose and Gln212, stabilizing substrate binding and reducing the K M value to approximately one-quarter of that of the wild type. Furthermore, a BLAST search with Lu-LGDH as a query resulted that homologs containing the glutamine residue at the corresponding position as well as other conserved catalytic and substrate-binding residues were found. With emerging studies on l -glucose biology, these results may contribute to enzymatic approaches for detecting the enzymes acting toward l -glucose, which is generally considered absent in nature.