Cloning of glutaryl-CoA dehydrogenase cDNA, and expression of wild type and mutant enzymes in Escherichia coli.
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Goodman SI, Kratz LE, DiGiulio KA, Biery BJ, Goodman KE, Isaya G, Frerman FE
Cloning of glutaryl-CoA dehydrogenase cDNA, and expression of wild type and mutant enzymes in Escherichia coli.
Hum Mol Genet. 1995 Sep;4(9):1493-8.
- PubMed ID
- 8541831 [ View in PubMed]
- Abstract
We have cloned, sequenced, and expressed cDNAs encoding wild type human glutaryl-CoA dehydrogenase subunit, and have expressed a mutant enzyme found in a patient with glutaric acidemia type I. The mutant protein is expressed at the same level as the wild type in Escherichia coli, but has less than 1% of the activity of wild-type dehydrogenase. We also present evidence that the glutaryl-CoA dehydrogenase transcript is alternatively spliced in human fibroblasts and liver; the alternatively spliced mRNA, when expressed in E.coli, encodes a stable but inactive protein. Purified expressed human glutaryl-CoA dehydrogenase has kinetic constants similar to those of the previously purified porcine dehydrogenase. The primary translation product from in vitro transcribed glutaryl-CoA dehydrogenase mRNA is translocated into mitochondria and processed in the same manner as most other nuclear-encoded mitochondrial proteins. Human glutaryl-CoA dehydrogenase shows 53% sequence similarity to porcine medium chain acyl-CoA dehydrogenase, and these similarities were utilized to predict structure-function relationships in glutaryl-CoA dehydrogenase.