Expression and assembly of mature apotransacylase (E(2b)) of bovine branched-chain α-keto acid dehydrogenase complex in Escherichia coli. Demonstration of transacylase activity and modification by lipoylation

Thomas A. Griffin, R. Max Wynn, David T. Chuang

Research output: Contribution to journalArticle

26 Scopus citations

Abstract

A cDNA clone encoding the entire transacylase (E(2b)) precursor of the bovine branched-chain α-keto acid dehydrogenase complex (Griffin, T.A., Lau, K.S., and Chuang, D.T. (1988) J. Biol. Chem. 263, 14008-14014) was used to construct a prokaryotic expression vector for recombinant mature E(2b). The overexpression in Escherichia coli correlates with the presence near the 5'-terminus of the mature E2b) coding region (nucleotides 20 to 28) of the seqauence 5'-TCAAACT-CT-3'. It has been proposed that this sequence is involved in secondary mRNA recognition through interaction with the 5'-terminus of the bacterial 16 S rRNA. The mature E(2b) protein has transacylase activity when assayed with exogenous dihydrolipoamide and [1-14C]isovaleryl-CoA as substrates. However, the recombinant protein has no attached lipoic acid. This was established by the absence of radiolabel incorporation when transformed E. coli cells were grown in a medium containing DL-[2-3H]lipoic acid. The recombinant mature E(2b) protein was purified to > 95% purity in one step using Sepharose 4B column chromatography. The purified recombinant protein was shown to have a cfubic 24-mer structure by electron microscopy and to possess a specific activity similar to that of the purified natural bovine E(2b). The purified recombinant mature E(2b) was lipoylated in vitro in the presence of 2 mM ATP using a mitochondrial extract prepared from bovine liver. The above results provide the first evidence that the proper folding and assembly of mature bovine E(2b) is independent of the attachment of lipoyl moieties and that mammalian lipoylation activity is present in mitochondria.

Original languageEnglish (US)
Pages (from-to)12104-12110
Number of pages7
JournalJournal of Biological Chemistry
Volume265
Issue number20
StatePublished - Aug 3 1990

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Expression and assembly of mature apotransacylase (E(2b)) of bovine branched-chain α-keto acid dehydrogenase complex in Escherichia coli. Demonstration of transacylase activity and modification by lipoylation'. Together they form a unique fingerprint.

  • Cite this