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

Most alanine dehydrogenases (AlaDHs), including that of Phormidium lapideum, a cyanobacterium, whose X-ray structure has been determined (PlaAlaDH, PDB entry: IPJC), 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 PIaAlaDH 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 Asp] 98 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. (C) 2004 Elsevier B.V. All rights reserved.