Gene. 86 (1990) 297-302 Elsevier GENE 03354 297 Cloning and eDNA sequence of the p-subunit component of human pyruvate dehydrogenase complex (Recombinant DNA; deduced primary amino acid sequence; inborn errors in pyruvate metabolism) Lap Ho and Mulehand S. Patel Department of Biochemistr),and Pew Center for Molecular Nutrition, Case Western Reserve UniversitySchool ofMedicine. Cleveland, OH 44106 (e.s.a.) Received by D.M. Skinner: 11 July 1989 Accepted: 28 August 1989 SUMMARY Two eDNA clones (ZEIpl, 1469 bp; ~Et/~2, 1437 bp) encoding the p-subunit of the pyruvate dehydrogenase (Et) component of the human pyruvate dehydrogenase complex were isolated from a human liver ~,gtll cDNA library. The composite human liver Etp eDNA encoded the entire mature EtP [329 amino acids (aa)] as well as a portion (26 aa) of the E~p leader peptide. Significant discrepancies were identified between the nucleotide and deduced aa sequences of human liver Em~ cDNAs and the corresponding sequences of a previously reported cultured human foreskin fibroblast E~/~ cDNA [Koike et al., Proc. Natl. Acad. Sci. USA 85 (1988) 41-45]. The composite human liver Etp cDNA generated in this study provides a reference sequence for investigating the structure-function relationship of human E~/~and for characterizing genetic mutations in patients with Et deficiency. INTRODUCTION The mammalian PDC is comprised of multiple copies of three catalytic component enzymes, namely pyruvate dehydrogenase (Et), dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase (Reed, 1974). El catalyzes the initial irreversible decarboxylation of pyruvate (Reed, 1974). It is a tetrameric enzyme composed of two copies each of an ~ and/~ subunit (Reed, 1974). It has been proposed that the Et • subunit catalyzes the pyruvate decar- Correspondence to: Dr. M.S. Patel, Department of Biochemistry, Case Western Reserve University School of Medicine, 2119 Abington Road, Cleveland, OH 44106 (U.S.A.) Tel. (216)368-3354; Fax (216)368-4544. Abbreviations: aa, amino acid(s); bp, base pair(s); ~Gal, p.gaiactosidase; eDNA, DNA complementary to RNA; Et, pyruvate dehydrogenase;Et, gene encoding Et; kb, kilobase(s) or 1000bp; nt, nucleotide(s); ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PDC, pyruvate dehydrogenase complex; SDS, sodium dodecyl sulfate; ss, single strand(ed). boxylation reaction while the subsequent reductive acetyla- tion of E2 iipoyl moieties is catalyzed by the EIP subunit (Roche and Reed, 1972; Hubner et al., 1978). There have been more than 100 reported cases of PDC deficiency. Analyses of PDC component enzymatic activities have established that the majority of PDC-deficient patients have low levels of Et activity only (Robinson et aft., 1987; Wexler et al., 1988). Since Et is a heterotetrameric enzyme and it is activated by a specific phospho-Et phosphatase (Reed, 1974), findings of low levels of Et activity in these patients could be due to defects of either the Et~ subunit, the Et~ subunit or the phospho-Et phosphatase. In isolated cases of Et deficiency, the diagnosis of specific E ~ defect has been based on finding of low levels of Et~ mRNA in the presence of normal levels of E ~ (Wexler et al., 1988). In the majority of cases of Et defects, however, it is not possible to identify mutations specific to either the Et~eor the Etp peptide by simple analysis of enzymatic activities, protein or mRNA profdes. The human Et~ gene has recently been identified on the X-chromosome (Brown 0378-11191901503.50 © 1990ElsevierSciencePublishers B.V.(BiomedicalDivision)