A conserved residue of L-alanine dehydrogenase from Bacillus pseudofirmus, Lys-73, participates in the catalytic reaction through hydrogen bonding

A multiple protein sequence alignment of L-alanine dehydrogenases from different bacterial species revealed that five highly conserved amino acid residues Arg-15, Lys-73, Lys-75, His-96 and Asp-269 are potential catalytic residues of L-alanine dehydrogenase from Bacillus pseudofirmus OF4. In this study, recombinant OF4Ald and its mutants of five conserved residues were constructed, expressed in Escherichia coil, purified by His(6)-tag affinity column and gel filtration chromatography, structure homology modeling, and characterized. The purified protein OF4Ald displayed high specificity to L-alanine (15 U mg(-1)) with an optimal temperature and pH of 40 degrees C and 10.5, respectively. Enzymatic assay and activity staining in native gels showed that mutations at four conserved residue Arg-15, Lys-75, His-96 and Asp-269 (except residue Lys-73) resulted in a complete loss in enzymatic activity, which signified that these predicted active sites are indispensable for OF4Ald activity. In contrast, the mutant K73A resulted in 6-fold improvement in k(cat)/K-m towards L-alanine as compared to the wild type protein. Further research of the residue Lys-73 substituted by various amino acids and structural modeling revealed that residue Lys-73 might be involved in the catalytic reaction of the enzyme by influencing the enzyme-substrate binding through the hydrogen-bonding interaction with conserved residue Lys-75.