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© 1995 Oxford University Press

RESEARCH-ARTICLE

Cloning of glutaryl-CoA dehydrogenase cDNA, and expression of wild type and mutant enzymes in Escherichia coli

Stephen I.Goodman*, Lisa E. Kratz1, Kathleen A. DiGiulio, Barbara J. Biery, Karen E. Goodman, Grazia Isaya2 and Frank E. Frerman

Departments of Pediatrics and Cellular and Structural Biology, University of Colorado School of Medicine Denver, CO. 1John F.Kennedy Center, Johns Hopkins School of Medicine, Baltimore 2MD and Department of Genetics, Yale University School of Medicine, New Haven, CT, USA

*To whom correspondence should be addressed

Received March 31, 1995; Accepted May 25, 1995

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.


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