Premature termination of transcription and alternative splicing in the human transacylase (E2) gene of the branched‐chain α‐ketoacid dehydrogenase complex

Abstract

We haw lsolat*:d ~t human =anemic clone hllF~ ~-,14 cont,ininll axons S, 6, 7 and 1~ or the branched.chain =.k=toa¢id deitydrolena=c E2 tran~a©)'htse l;ene. Sequencinj ofcxon It and its surroandinlt introl~ic ~equcnc©s reveals complct© identity with th~ previously reported truncated E2 eDNA thE2. I) sequence between nu~:l¢otid=s g],tl ttnd 1521, We have identified ¢oflNnsus splice= cite junctions ntankinj exon I~ and trite a cryptic 3" splice site 370 ba~¢s up~ireaTn from the start of exon 14 in the sane, In addition, two polyadenyl~tign sililnals Iocatedl in the hE2. I eDNA are also present in the intronic sequence downslremn of'exert 8 which promote =©rmiaation or transeriplion. The data indicate that shortel'~:d human liver E! transcripts underlie alternative =plicinlt to ~¢idd rnRNA orthe hE2.1 type, Genomic clone; eDNA" E2 gear; Alternative splieinil 1, INTRODUCTIONThe branched chain a-kate acid dehydrogenas¢ complex (abbreviated branched chain complex) catalyzes the rate-limiting step in the oxidative decarboxylation of the branched-chain c~-keto acids derived from leucine, isoleucin¢ and valine, It is a mitochondrial multienzyme complex that is both structurally and mechanistically analogous to the pyruvat¢ and ~-ketoglutarate dehydrogenase complexes [1]. The branched-chain corn plex consists of three catalytic components: a branched-chain c~-keto acid decarboxylase or El, a dihydrolipoyl transacylase or E2 and a dihydrolipoyl dehydrogenase or E3 [2,3]. The E3 component is common to all three a-kate acid dehydrogenase complexes., The branched-chain complex also has two regulatory enzymes, a specific kinase and a specific phosphatase [4][5][6][7][8]. The E2 subunit forms a 24-mar cubic core to which El, E3, the kinase and the phosphatase are attached through non-covalent interactions. Studies on the human and bovine E2 subunits have revealed that the E2 subunit contains three distinct domains: a lipoyl-bearing domain, an E3-binding domain and an inner-core domain [9,10], Similar E2 domain structures are also found in other c~-keto acid dehydrogenase complexes from both eukaryotic and prokaryotic organisms [11,12] We and others [10,I3] have previously isolated a human liver E2 cDNA clone, This clone (hE2-t) is 1577 bp and contains a poly(A)+ tail preceded by 2 polyadenylation signals [10], The amino acid sequence predicted by its nucleotide sequence codes for the lipoyl-bearing and E3 binding domains of the E2 polypeptide; however, it lacks the inner.core domain of the E2 polypeptide as described later by the isolation of the full-length hum'." 72 eDNA [14], During the course of cloning the hun,..a E2 dane, we isolated a genomic clone (huE2-14) that contains intronic sequences flankmg the eighth exon of the E2 gene which are identical to the nucleotid¢ sequences of hE2-1 bases 938'1521, Within this generate region is exert 8 consisting of 78 bp coding for Asp-253 to Lys-278 (Lau and Chuang, unpublished results). In addition, two polyadenylation signals are present downstream of exon 8. We propose that prem...

Fourteen new mutations of BCKDHA, BCKDHB and DBT genes associated with maple syrup urine disease (MSUD) in Malaysian population

“…The E2b subunits of BCKDC contains an N-terminal lipoyl-bearing domain (LBD), an internal subunit-binding domain (SBD) and a C-terminal core (catalytic) domain (E2bCD) [ 21 , 22 ] . Taking into account the protein sequence of bovine shares 93% identity with human homology and the identified mutation occurs in E2bCD domain, the bovine protein structure was used as a model to study the effects of MSUD mutation in human.…”

A synchronized substrate-gating mechanism revealed by cubic-core structure of the bovine branched-chain α-ketoacid dehydrogenase complex

“…The E2b subunit of BCKDC consists of an N‐terminal lipoyl‐bearing domain (LBD), an internal subunit‐binding domain (SBD) and a C‐terminal core (catalytic) domain (E2bCD) (Griffin et al , 1988; Lau et al , 1991). The E2bCD possesses a catalytic site for the acyltransfer reaction and is responsible for the formation of the cubic core.…”