Role of residue F180 on the coenzyme selectivity of E. coli lactaldehyde dehydrogenase

Aldehyde dehydrogenases (ALDHs) catalyze the conversion of aldehydes to their corresponding acids by the reduction of NAD(P) + . ALDHs have been related to the unspecific detoxification of aldehydes, but some ALDHs have specific roles in metabolic pathways. Phenylacetaldehyde dehydrogenase (PAD) and lactaldehyde dehydrogenase (ALD) from E. coli , are involved in the catabolism of L‐phenylalanine and L‐fucose, respectively. They share some structural and kinetic properties. One main difference between these enzymes is that PAD can use NAD + and NADP + , while ALD only uses NAD + . In 10 residues around E179 (proposed to be important for the exclusion of NADP + in NAD + ‐dependent dehydrogenases), PAD and ALD are identical except by amino acid 180, which is T in PAD and F in ALD. Thus, we hypothesized that T180 exerts a steric effect impairing the binding of NADP + to ALD. Mutation of F180 to T rendered an enzyme with the ability to bind and use NADP + . This was shown by studies of protection against thermal denaturalization and by activity. ALDF180T exhibited a Km and Vm for NADP + of 78 μM and 0.87 μmol/min*mg, respectively. This activity was higher than that of ALD wild type with NAD + . Further, ALD‐F180T showed a 18‐fold increment of Vm/Km ratio with NAD + compared to ALD wild type. In conclusion, residue F180 is exerting a steric effect on the coenzyme binding site, impairing not only the binding of NADP + , but also the correct binding of NAD +