TY - JOUR
T1 - Cloning of glutaryl-coa dehydrogenase cDNA, and expression of wild type and mutant enzymes in Escherichia coli
AU - I.goodman, Stephen
AU - Kratz, Lisa E.
AU - Digiulio, Kathleen A.
AU - Biery, Barbara J.
AU - Goodman, Karen E.
AU - Isaya, Grazia
AU - Frerman, Frank E.
N1 - Funding Information:
We thank Drs Jung-Ja Kim, Medical College of Wisconsin, and Gerald Vockley, Mayo Clinic, for very helpful discussions. This work was supported in part by grants HD 08313, HD 04024 and GM 48076 (to G.I.) from the National Institutes of Health.
PY - 1995/9
Y1 - 1995/9
N2 - 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.
AB - 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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U2 - 10.1093/hmg/4.9.1493
DO - 10.1093/hmg/4.9.1493
M3 - Article
C2 - 8541831
AN - SCOPUS:0029084073
SN - 0964-6906
VL - 4
SP - 1493
EP - 1498
JO - Human molecular genetics
JF - Human molecular genetics
IS - 9
ER -