Human liver mitochondrial aldehyde dehydrogenase: Three-dimensional structure and the restoration of solubility and activity of chimeric forms

Human liver cytosolic and mitochondrial isozymes of aldehyde dehydrogenase share 70% sequence identity. However, the first 21 residues are not conserv ed between the human isozymes (15% identity). The three-dimensional structu res of the beef mitochondrial and sheep cytosolic forms have virtually iden tical three-dimensional structures. Here, we solved the structure of the hu man mitochondrial enzyme and found it to be identical to the beef enzyme. T he first 21 residues are found on the surface of the enzyme and make no con tact with other subunits in the tetramer A pair of chimeric enzymes between the human isozymes was made. Each chimera had the first 21 residues from o ne isozyme and the remaining 479 from the other. When the first 21 residues were from the mitochondrial isozyme, an enzyme with cytosolic-like propert ies was produced. The other was expressed but was insoluble. It was possibl e to restore solubility and activity to the chimera that had the first 21 c ytosolic residues fused to the mitochondrial ones by making point mutations to residues at the N-terminal end. When residue 19 was changed from tyrosi ne to a cysteine, the residue found in the mitochondrial form, an active en zyme could be made though the K-m for NAD(+) was 35 times higher than the n ative mitochondrial isozyme and the specific activity was reduced by 75%. T his residue interacts with residue 203, a nonconserved, nonactive site resi due. A mutation of residue 18, which also interacts with 203, restored solu bility, but not activity. Mutation to residue 15, which interacts with 104, also restored solubility but not activity. It appears that to have a solub le or active enzyme a favorable interaction must occur between a residue in a surface loop and a residue elsewhere in the molecule even though neither make contact with the active site region of the enzyme.