title: Expression and assembly of a functional E1 component (alpha 2 beta 2) of mammalian branched-chain alpha-ketoacid dehydrogenase complex in Escherichia coli.
abstract: We have expressed an active recombinant E1 decarboxylase component of the mammalian branched-chain alpha-ketoacid dehydrogenase complex in Escherichia coli by subcloning mature E1 alpha and E1 beta subunit cDNA sequences into a bacterial expression vector. To permit affinity purification under native conditions, the mature E1 alpha subunit was fused with the affinity ligand E. coli maltose-binding protein (MBP) through an endoprotease Factor Xa-specific linker peptide. When co-expressed, the MBP-E1 alpha fusion and E1 beta subunits were shown to co-purify as a MBP-E1 component that exhibited both E1 activity and binding competence for recombinant branched-chain E2 component. In contrast, in vitro mixing of individually expressed MBP-E1 alpha and E1 beta did not result in assembly or produce E1 activity. Following proteolytic removal of the affinity ligand and linker peptide with Factor Xa, a recombinant E1 species was eluted from a Sephacryl S-300HR sizing column as an enzymatically active 160-kDa species. The latter showed 1:1 subunit stoichiometry, which was consistent with an alpha 2 beta 2 structure. The recovery of this 160-kDa recombinant E1 species (estimated at 0.07% of total lysate protein) was low, with the majority of the recombinant protein lost as insoluble aggregates. Our findings suggest that the concurrent expression of both E1 alpha and E1 beta subunits in the same cellular compartment is important for assembly of both subunits into a functional E1 alpha 2 beta 2 heterotetramer. By using this co-expression system, we also find that the E1 alpha missense mutation (Tyr-393----Asn) characterized in Mennonites with maple syrup urine disease prevents the assembly of soluble E1 heterotetramers.
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235-9038.
authors.forename: J R
authors.initials: JR
authors.lastname: Davie
authors.forename: R M
authors.initials: RM
authors.lastname: Wynn
authors.forename: R P
authors.initials: RP
authors.lastname: Cox
authors.forename: D T
authors.initials: DT
authors.lastname: Chuang
mesh_terms: D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D002417:Cattle; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D015951:Factor Xa; D005822:Genetic Vectors; D006801:Humans; D007658:Ketone Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D046911:Macromolecular Substances; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008375:Maple Syrup Urine Disease / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification
chemical_list: D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009097:Multienzyme Complexes; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D008320:Maltose; D004247:DNA; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D015951:Factor Xa