A single amino acid mutation alters substrate specificity towards l-glucose in a bacterial l-galactose dehydrogenase.

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.