Structure and selectivity in post-translational modification: attaching the biotinyl–lysine and lipoyl–lysine swinging arms in multifunctional enzymes

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Abstract
The post‐translational attachment of biotin and lipoic acid to specific lysine residues displayed in protruding β‐turns in homologous biotinyl and lipoyl domains of their parent enzymes is catalysed by two different ligases. We have expressed in Escherichia coli a sub‐gene encoding the biotinyl domain of E.coli acetyl‐CoA carboxylase, and by a series of mutations converted the protein from the target for biotinylation to one for lipoylation, in vivo and in vitro . The biotinylating enzyme, biotinyl protein ligase (BPL), and the lipoylating enzyme, LplA, exhibited major differences in the recognition process. LplA accepted the highly conserved MKM motif that houses the target lysine residue in the biotinyl domain β‐turn, but was responsive to structural cues in the flanking β‐strands. BPL was much less sensitive to changes in these β‐strands, but could not biotinylate a lysine residue placed in the DKA motif characteristic of the lipoyl domain β‐turn. The presence of a further protruding thumb between the β2 and β3 strands in the wild‐type biotinyl domain, which has no counterpart in the lipoyl domain, is sufficient to prevent aberrant lipoylation in E.coli . The structural basis of this discrimination contrasts with other forms of post‐translational modification, where the sequence motif surrounding the target residue can be the principal determinant.

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