Conversion of cofactor specificities of alanine dehydrogenases by site-directed mutagenesis

Abstract

Most alanine dehydrogenases (AlaDHs), including that of Phormidium lapideum, a cyanobacterium, whose X-ray structure has been determined (PlaAlaDH, PDB entry: 1PJC), are strictly NAD +-specific. However, AlaDH from a psychrophile, Shewanella sp. Ac10 (SheAlaDH), exhibits dual specificity for NAD + and NADP +. As Ile198 at the cofactor-binding site of PlaAlaDH is replaced by a corresponding arginine (Arg199) in SheAlaDH, we speculate that this arginine residue may serve as the binding site for the 2′-phosphate group of NADP + in SheAlaDH. To verify this speculation, Arg199 of SheAlaDH was replaced by isoleucine via site-directed mutagenesis, and the resulting mutant enzyme (Arg199Ile) indeed was shown to act specifically on NAD +. On the other hand, Ile198Arg mutant of PlaAlaDH acted not only on NAD + but also on NADP +. It was also observed that Asp197 of PlaAlaDH is conserved among various NAD +-specific amino acid dehydrogenases, including SheAlaDH, but is replaced by hydrophobic amino acids in NADP +-specific enzymes. To investigate the relevance of this residue, mutant SheAlaDHs with Asp198 replaced by either Gly, Ala, Val or Leu were obtained. The k cat/ K m values for NADP + were increased from 5- to 270-folds by the mutation with Asp198Ala being the best catalyst. Thus, AlaDHs with modified coenzyme specificities can be obtained by single site-specific mutations.