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Title:Regulatory function and interdomain communication of group II biotin protein ligases
Author(s):Henke, Sarah K
Director of Research:Cronan, John E.
Doctoral Committee Chair(s):Cronan, John E.
Doctoral Committee Member(s):Gardner, Jeffrey F.; Imlay, James A.; Metcalf, William W.
Department / Program:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Biotin protein ligase
Transcriptional regulation
Abstract:Biotin is a cofactor required for function of essential biotin dependent enzymes that are involved in metabolic processes including fatty acid biosynthesis, gluconeogenesis, and amino acid catabolism. In order for biotin to act as a cofactor it must first be covalently attached to the biotin dependent enzyme. Biotin protein ligase (BPL) catalyzes this covalent attachment in a two-step reaction. First BPL binds biotin and ATP to synthesize the intermediate biotinoyl-AMP, releasing pyrophosphate. In the second half reaction a conserved lysine residue from the biotin dependent enzyme acts as a nucleophile and attacks the mixed anhydride of biotinoyl-AMP to give the biotinylated protein and release of AMP. Microbial BPLs are classified into two groups. Both group I and group II BPLs have catalytic cores and C-terminal domains that are well conserved between the groups. Group II biotin protein ligases are characterized by the presence of an N-terminal DNA binding domain that functions in transcriptional regulation of the genes of biotin biosynthesis. Escherichia coli BPL, called BirA, is the best-studied example of how an enzyme can also act as a transcriptional regulator of the biotin biosynthetic operon. Transcriptional repression of the E. coli biotin operon occurs when biotin acceptor proteins (biotin dependent enzymes) have been fully biotinylated thereby allowing BirA to accumulate biotinoyl-AMP in its active site. The presence of biotinoyl-AMP results in BirA dimerization and subsequent DNA binding that represses transcription of the biotin biosynthetic operon. Derepression of the biotin operon transcription occurs upon low biotin levels or increased levels of unmodified biotin acceptor proteins. Transfer of accumulated biotinoyl-AMP to acceptor proteins results in monomeric BirA which is unable to bind the biotin operator. The N-terminal DNA binding domain of E. coli BirA is separated from the central domain by a linker region. It was proposed that E. coli BirA could be transformed into a group I BPL by simply removing the N-terminal DNA binding domain, where it would no longer have regulatory activity but would retain ligase activity. However, the E. coli BirA ligase activity was severely compromised when the N-terminal domain was removed, suggesting interdomain communication is required between the DNA binding domain and the central domain for normal ligase activity. The Bacillus subtilis BPL, BirA, is also 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. The Staphylococcus aureus Group II BPL, BirA, has been reported to bind an imperfect inverted repeat located upstream of the biotin synthesis operon. DNA binding by other Group II BPLs requires dimerization of the protein, which is triggered by synthesis of biotinoyl-AMP (biotinoyl-adenylate), the intermediate in the ligation of biotin to its cognate target proteins. However, the S. aureus BirA was reported to dimerize and bind DNA in the absence of biotin or biotinoyl-AMP (85). These in vitro results argued that the protein would be unable to respond to the levels of biotin or acceptor proteins and thus would lack the regulatory properties of the other characterized BirA proteins. We tested the regulatory function of the S. aureus protein using an in vivo model system and examined its DNA binding properties in vitro using electrophoretic mobility shift and fluorescence anisotropy analyses. We report that the S. aureus BirA is an effective regulator of biotin operon transcription and that the prior data can be attributed to artifacts of mobility shift analyses. We also report that deletion of the DNA binding domain of the S. aureus BirA results in loss of virtually all of its ligation activity.
Issue Date:2017-08-24
Rights Information:Copyright 2017 Sarah Henke
Date Available in IDEALS:2018-03-13
Date Deposited:2017-12

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