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

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 E_2b coding region (nucleotides 20 to 28) of the sequence 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 cubic 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.