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 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.
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Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA.
Zhou
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J
Hurley
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TD
Weiner
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D000444:Aldehyde Dehydrogenase / Q000737:chemistry*; D018360:Crystallography, X-Ray; D005453:Fluorescence; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry; D008930:Mitochondria, Liver / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012995:Solubility
D007527:Isoenzymes; D000444:Aldehyde Dehydrogenase