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Successful conversion of the Bacillus subtilis BirA Group II biotin protein ligase into a Group I ligase.

Henke SK, Cronan JE - PLoS ONE (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Illinois, Urbana, Illinois, United States of America.

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.

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Chemical crosslinking of the B. subtilis wild type and Δ2-65 BirA proteins.A. Wild type BirA. B. The Δ2-65 BirA. Note that efficient dimer formation requires the presence of both biotin and ATP. The EGS concentrations are in mM.
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pone-0096757-g009: Chemical crosslinking of the B. subtilis wild type and Δ2-65 BirA proteins.A. Wild type BirA. B. The Δ2-65 BirA. Note that efficient dimer formation requires the presence of both biotin and ATP. The EGS concentrations are in mM.

Mentions: The ligase activities of the purified B. subtilis BirA N-terminal deletion proteins (Fig. 3) were also tested by in vitro biotinylation assays. As expected from the complementation results the Δ2-63 and Δ2-65 BirA proteins showed Bio-5′-AMP synthesis and biotin transfer activities indistinguishable from those of the wild type protein (Fig. 8). In contrast the Bio-5′-AMP synthetic abilities of the Δ2-74 and Δ1-81 BirAs were significantly reduced relative to wild type BirA. However, the biotin transfer activity of the Δ2-74 BirA was comparable to wild type levels. Biotin transfer by the Δ1-81 BirA was significantly reduced compared to the wild type protein (Fig. 9). In agreement with prior work [16], [17] the E. coli Δ2-65 BirA was significantly reduced in Bio-5′-AMP synthesis and in biotin transfer (Fig. 8).


Successful conversion of the Bacillus subtilis BirA Group II biotin protein ligase into a Group I ligase.

Henke SK, Cronan JE - PLoS ONE (2014)

Chemical crosslinking of the B. subtilis wild type and Δ2-65 BirA proteins.A. Wild type BirA. B. The Δ2-65 BirA. Note that efficient dimer formation requires the presence of both biotin and ATP. The EGS concentrations are in mM.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016012&req=5

pone-0096757-g009: Chemical crosslinking of the B. subtilis wild type and Δ2-65 BirA proteins.A. Wild type BirA. B. The Δ2-65 BirA. Note that efficient dimer formation requires the presence of both biotin and ATP. The EGS concentrations are in mM.
Mentions: The ligase activities of the purified B. subtilis BirA N-terminal deletion proteins (Fig. 3) were also tested by in vitro biotinylation assays. As expected from the complementation results the Δ2-63 and Δ2-65 BirA proteins showed Bio-5′-AMP synthesis and biotin transfer activities indistinguishable from those of the wild type protein (Fig. 8). In contrast the Bio-5′-AMP synthetic abilities of the Δ2-74 and Δ1-81 BirAs were significantly reduced relative to wild type BirA. However, the biotin transfer activity of the Δ2-74 BirA was comparable to wild type levels. Biotin transfer by the Δ1-81 BirA was significantly reduced compared to the wild type protein (Fig. 9). In agreement with prior work [16], [17] the E. coli Δ2-65 BirA was significantly reduced in Bio-5′-AMP synthesis and in biotin transfer (Fig. 8).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Illinois, Urbana, Illinois, United States of America.

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.

Show MeSH