Abstract
Group II biotin protein ligases (BPLs) are characterized by the presence of an N-terminal DNA binding domain that allows transcriptional regulation of biotin biosynthetic and transport genes whereas Group I BPLs lack this N-terminal domain. The Bacillus subtilis BPL, BirA, is classified as a Group II BPL based on sequence predictions of an N-terminal helix-turn-helix motif and mutational alteration of its regulatory properties. We report evidence that B. subtilis BirA is a Group II BPL that regulates transcription at three genomic sites: bioWAFDBI, yuiG and yhfUTS. Moreover, unlike the paradigm Group II BPL, E. coli BirA, the N-terminal DNA binding domain can be deleted from Bacillus subtilis BirA without adverse effects on its ligase function. This is the first example of successful conversion of a Group II BPL to a Group I BPL with retention of full ligase activity.
Highlights
Biotin protein ligase (BPL) is required for the covalent attachment of biotin to biotin-dependent enzymes
Full dependence of binding on ATP and biotin required treatment of the protein with neutral hydroxylamine to remove Bio-59-AMP accumulated in the active site during expression in E. coli
BirA did not interact with a site that was composed of only one of the B. subtilis biosynthetic operator (bioO) inverted repeats suggesting that the form of BirA active in DNA binding is a dimer (Fig. 4H)
Summary
Biotin protein ligase (BPL) is required for the covalent attachment of biotin to biotin-dependent enzymes. In all four E. coli BirA crystal structures [13,14,15] the HTH structure is spatially well removed from the other domains of the protein and deletion of the N-terminal DNA binding domain was expected to convert this Group II BPL into a fully functional Group I ligase. This was not the case: the resulting protein had severely compromised ligase activity [16]. This was true for ligases having smaller N-terminal deletions [17]
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