Human liver cytosolic and mitochondrial isozymes
of aldehyde dehydrogenase share 70% sequence identity.
However, the first 21 residues are not conserved between
the human isozymes (15% identity). The three-dimensional
structures of the beef mitochondrial and sheep cytosolic
forms have virtually identical three-dimensional structures.
Here, we solved the structure of the human mitochondrial
enzyme and found it to be identical to the beef enzyme.
The first 21 residues are found on the surface of the enzyme
and make no contact 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 one isozyme
and the remaining 479 from the other. When the first 21
residues were from the mitochondrial isozyme, an enzyme
with cytosolic-like properties was produced. The other
was expressed but was insoluble. It was possible to restore
solubility and activity to the chimera that had the first
21 cytosolic residues fused to the mitochondrial ones by
making point mutations to residues at the N-terminal end.
When residue 19 was changed from tyrosine to a cysteine,
the residue found in the mitochondrial form, an active
enzyme could be made though the Km
for NAD+ was 35 times higher than the native
mitochondrial isozyme and the specific activity was reduced
by 75%. This residue interacts with residue 203, a nonconserved,
nonactive site residue. A mutation of residue 18, which
also interacts with 203, restored solubility, but not activity.
Mutation to residue 15, which interacts with 104, also
restored solubility but not activity. It appears that to
have a soluble 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.