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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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The biotin protein ligase (BPL) reaction.Attachment of biotin to acceptor proteins occurs in a two-step reaction. First, BirA binds biotin and ATP to synthesize Bio-5′-AMP (biotinoyl-5'-adenylate) with release of pyrophosphate. In the second step the conserved lysine residue of the acceptor protein acts as a nucleophile and attacks the mixed anhydride bond to give the biotinylated acceptor protein plus AMP.
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pone-0096757-g001: The biotin protein ligase (BPL) reaction.Attachment of biotin to acceptor proteins occurs in a two-step reaction. First, BirA binds biotin and ATP to synthesize Bio-5′-AMP (biotinoyl-5'-adenylate) with release of pyrophosphate. In the second step the conserved lysine residue of the acceptor protein acts as a nucleophile and attacks the mixed anhydride bond to give the biotinylated acceptor protein plus AMP.

Mentions: Biotin protein ligase (BPL) is required for the covalent attachment of biotin to biotin-dependent enzymes. This attachment proceeds in a two-step reaction. First, BPL binds both biotin and ATP to synthesize biotinoyl-5′-AMP (Bio-5′-AMP, also called biotinoyl-adenylate) with release of pyrophosphate [1]. The ε-amino group of the conserved lysine residue of the acceptor protein acts as a nucleophile to attack the Bio-5′-AMP mixed anhydride bond to give covalently attached biotin plus AMP (Fig. 1). Microbial BPLs are readily placed into two groups [2]. Both groups have catalytic and C-terminal domains that show strong structural conservation [3]–[7] whereas Group II BPLs are characterized by addition of an N-terminal helix-turn-helix (HTH) DNA binding domain that permits transcriptional regulation of the biotin synthetic genes. E. coli BirA, the paradigm for regulation of biotin biosynthesis, is the best studied Group II BPL. Transcriptional repression of the E. coli biotin operon occurs when BirA accumulates Bio-5′-AMP because all biotin acceptor proteins have been biotinylated [8]–[10]. Bio-5′-AMP accumulation results in dimerization of BirA and subsequent DNA binding [6], [11], [12]. In all four E. coli BirA crystal structures [13]–[15] the HTH structure is spatially well removed from the other domains of the protein and thus deletion of the N-terminal DNA binding domain was expected to convert this Group II BPL into a fully functional Group I ligase. However, this was not the case: the resulting protein had severely compromised ligase activity [16]. This was also true for ligases having smaller N-terminal deletions [17].


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

Henke SK, Cronan JE - PLoS ONE (2014)

The biotin protein ligase (BPL) reaction.Attachment of biotin to acceptor proteins occurs in a two-step reaction. First, BirA binds biotin and ATP to synthesize Bio-5′-AMP (biotinoyl-5'-adenylate) with release of pyrophosphate. In the second step the conserved lysine residue of the acceptor protein acts as a nucleophile and attacks the mixed anhydride bond to give the biotinylated acceptor protein plus AMP.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0096757-g001: The biotin protein ligase (BPL) reaction.Attachment of biotin to acceptor proteins occurs in a two-step reaction. First, BirA binds biotin and ATP to synthesize Bio-5′-AMP (biotinoyl-5'-adenylate) with release of pyrophosphate. In the second step the conserved lysine residue of the acceptor protein acts as a nucleophile and attacks the mixed anhydride bond to give the biotinylated acceptor protein plus AMP.
Mentions: Biotin protein ligase (BPL) is required for the covalent attachment of biotin to biotin-dependent enzymes. This attachment proceeds in a two-step reaction. First, BPL binds both biotin and ATP to synthesize biotinoyl-5′-AMP (Bio-5′-AMP, also called biotinoyl-adenylate) with release of pyrophosphate [1]. The ε-amino group of the conserved lysine residue of the acceptor protein acts as a nucleophile to attack the Bio-5′-AMP mixed anhydride bond to give covalently attached biotin plus AMP (Fig. 1). Microbial BPLs are readily placed into two groups [2]. Both groups have catalytic and C-terminal domains that show strong structural conservation [3]–[7] whereas Group II BPLs are characterized by addition of an N-terminal helix-turn-helix (HTH) DNA binding domain that permits transcriptional regulation of the biotin synthetic genes. E. coli BirA, the paradigm for regulation of biotin biosynthesis, is the best studied Group II BPL. Transcriptional repression of the E. coli biotin operon occurs when BirA accumulates Bio-5′-AMP because all biotin acceptor proteins have been biotinylated [8]–[10]. Bio-5′-AMP accumulation results in dimerization of BirA and subsequent DNA binding [6], [11], [12]. In all four E. coli BirA crystal structures [13]–[15] the HTH structure is spatially well removed from the other domains of the protein and thus deletion of the N-terminal DNA binding domain was expected to convert this Group II BPL into a fully functional Group I ligase. However, this was not the case: the resulting protein had severely compromised ligase activity [16]. This was also true for ligases having smaller N-terminal deletions [17].

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