Aldehyde dehydrogenase - Maintaining critical active site geometry at motif 8 in the class 3 enzyme

Alignment of all known, diverse members of the aldehyde dehydrogenase (ALDH ) extended family revealed only two strictly conserved, nonglycine residues , a glutamate and a phenylalanine residue. Both occur in one of the highly conserved 'motif' segments and both occupy strategic locations in the terti ary structure at the bottom of the catalytic funnel. In class 3 ALDH, these are Glu333 and Phe335. In addition, Asp247, which is not highly conserved but is characteristic of class 3 ALDHs, hydrogen bonds the main chain betwe en Glu333 and Phe335. These three residues were mutated conservatively. Mic haelis constants determined for both NAD/propanal and NADP/benzaldehyde sub strate pairs show all three residues to be crucial to effective catalysis, and suggest that the hydrogen bond to Asp247 is a key element in maintainin g precise geometry of key elements at the active site.