Overexpression of human antiquitin in E. coli: enzymatic characterization of twelve ALDH7A1 missense mutations associated with pyridoxine-dependent epilepsy.
Pyridoxine dependent epilepsy is an autosomal recessive disorder characterized by early onset seizures responsive to pyridoxine and caused by a defect in the α-aminoadipic semialdehyde dehydrogenase (antiquitin) gene (ALDH7A1). In order to characterize the effects of a series of twelve disease-associated ALDH7A1 missense mutations on antiquitin activity, we generated the mutations in a recombinant human antiquitin cDNA and expressed them in Escherichia coli. We developed an automated spectrophotometric assay of antiquitin enzymatic activity using the natural substrate α-aminoadipic semialdehyde. The substrate was generated using a recombinant lysine aminotransferase gene (lat) from Streptomyces clavuligerus. In the E. coli expression system all the mutants were stably expressed but lacked enzymatic activity. This is consistent with pathogenicity of these mutations in vivo.
Department of Pediatrics, University of British Columbia, Vancouver, BC, Canada. marioncm@mail.ubc.ca
Marion B
MB
Coulter-Mackie
Ailin
A
Li
Qun
Q
Lian
Eduard
E
Struys
Sylvia
S
Stockler
Paula J
PJ
Waters
D000444:Aldehyde Dehydrogenase / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D057075:Enzyme Assays; D004827:Epilepsy / Q000201:enzymology* / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D050505:Mutant Proteins / Q000378:metabolism; D020125:Mutation, Missense / Q000235:genetics*; D013997:Time Factors
D050505:Mutant Proteins; C089507:ALDH7A1 protein, human; D000444:Aldehyde Dehydrogenase