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Ligand specificity of group I biotin protein ligase of Mycobacterium tuberculosis.

Purushothaman S, Gupta G, Srivastava R, Ramu VG, Surolia A - PLoS ONE (2008)

Bottom Line: MtBPL forms 11 H-bonds with biotin, relative to 35 with bio-5'AMP.The enzyme catalyzed transfer of biotin to BCCP was confirmed by incorporation of radioactive biotin and by Avidin blot.MtBPL has low affinity (K(b) = 1.06x10(-6) M) for biotin relative to EcBirA but their K(m) are almost comparable suggesting that while the major function of MtBPL is biotinylation of BCCP, tight binding of biotin/bio-5'AMP by EcBirA is channeled for its repressor activity.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.

ABSTRACT

Background: Fatty acids are indispensable constituents of mycolic acids that impart toughness & permeability barrier to the cell envelope of M. tuberculosis. Biotin is an essential co-factor for acetyl-CoA carboxylase (ACC) the enzyme involved in the synthesis of malonyl-CoA, a committed precursor, needed for fatty acid synthesis. Biotin carboxyl carrier protein (BCCP) provides the co-factor for catalytic activity of ACC.

Methodology/principal findings: BPL/BirA (Biotin Protein Ligase), and its substrate, biotin carboxyl carrier protein (BCCP) of Mycobacterium tuberculosis (Mt) were cloned and expressed in E. coli BL21. In contrast to EcBirA and PhBPL, the approximately 29.5 kDa MtBPL exists as a monomer in native, biotin and bio-5'AMP liganded forms. This was confirmed by molecular weight profiling by gel filtration on Superdex S-200 and Dynamic Light Scattering (DLS). Computational docking of biotin and bio-5'AMP to MtBPL show that adenylation alters the contact residues for biotin. MtBPL forms 11 H-bonds with biotin, relative to 35 with bio-5'AMP. Docking simulations also suggest that bio-5'AMP hydrogen bonds to the conserved 'GRGRRG' sequence but not biotin. The enzyme catalyzed transfer of biotin to BCCP was confirmed by incorporation of radioactive biotin and by Avidin blot. The K(m) for BCCP was approximately 5.2 microM and approximately 420 nM for biotin. MtBPL has low affinity (K(b) = 1.06x10(-6) M) for biotin relative to EcBirA but their K(m) are almost comparable suggesting that while the major function of MtBPL is biotinylation of BCCP, tight binding of biotin/bio-5'AMP by EcBirA is channeled for its repressor activity.

Conclusions/significance: These studies thus open up avenues for understanding the unique features of MtBPL and the role it plays in biotin utilization in M. tuberculosis.

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Flourescence titration.Steady state fluorescence emission spectra of MtBPL in absence and presence of biotin. [BPL] = 2 µM, [biotin] = 50 µM. All spectra were obtained in standard buffer at 20°C.
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pone-0002320-g005: Flourescence titration.Steady state fluorescence emission spectra of MtBPL in absence and presence of biotin. [BPL] = 2 µM, [biotin] = 50 µM. All spectra were obtained in standard buffer at 20°C.

Mentions: Fluorescence titration of BPL: Figure 5 shows steady state fluorescence emission spectra of MtBPL, MtBPL + biotin. The emission maximum for MtBPL was at 342 nm similar to EcBirA. The binding of biotin to the protein resulted in 10% quenching of intrinsic fluorescence signal.


Ligand specificity of group I biotin protein ligase of Mycobacterium tuberculosis.

Purushothaman S, Gupta G, Srivastava R, Ramu VG, Surolia A - PLoS ONE (2008)

Flourescence titration.Steady state fluorescence emission spectra of MtBPL in absence and presence of biotin. [BPL] = 2 µM, [biotin] = 50 µM. All spectra were obtained in standard buffer at 20°C.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2384007&req=5

pone-0002320-g005: Flourescence titration.Steady state fluorescence emission spectra of MtBPL in absence and presence of biotin. [BPL] = 2 µM, [biotin] = 50 µM. All spectra were obtained in standard buffer at 20°C.
Mentions: Fluorescence titration of BPL: Figure 5 shows steady state fluorescence emission spectra of MtBPL, MtBPL + biotin. The emission maximum for MtBPL was at 342 nm similar to EcBirA. The binding of biotin to the protein resulted in 10% quenching of intrinsic fluorescence signal.

Bottom Line: MtBPL forms 11 H-bonds with biotin, relative to 35 with bio-5'AMP.The enzyme catalyzed transfer of biotin to BCCP was confirmed by incorporation of radioactive biotin and by Avidin blot.MtBPL has low affinity (K(b) = 1.06x10(-6) M) for biotin relative to EcBirA but their K(m) are almost comparable suggesting that while the major function of MtBPL is biotinylation of BCCP, tight binding of biotin/bio-5'AMP by EcBirA is channeled for its repressor activity.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.

ABSTRACT

Background: Fatty acids are indispensable constituents of mycolic acids that impart toughness & permeability barrier to the cell envelope of M. tuberculosis. Biotin is an essential co-factor for acetyl-CoA carboxylase (ACC) the enzyme involved in the synthesis of malonyl-CoA, a committed precursor, needed for fatty acid synthesis. Biotin carboxyl carrier protein (BCCP) provides the co-factor for catalytic activity of ACC.

Methodology/principal findings: BPL/BirA (Biotin Protein Ligase), and its substrate, biotin carboxyl carrier protein (BCCP) of Mycobacterium tuberculosis (Mt) were cloned and expressed in E. coli BL21. In contrast to EcBirA and PhBPL, the approximately 29.5 kDa MtBPL exists as a monomer in native, biotin and bio-5'AMP liganded forms. This was confirmed by molecular weight profiling by gel filtration on Superdex S-200 and Dynamic Light Scattering (DLS). Computational docking of biotin and bio-5'AMP to MtBPL show that adenylation alters the contact residues for biotin. MtBPL forms 11 H-bonds with biotin, relative to 35 with bio-5'AMP. Docking simulations also suggest that bio-5'AMP hydrogen bonds to the conserved 'GRGRRG' sequence but not biotin. The enzyme catalyzed transfer of biotin to BCCP was confirmed by incorporation of radioactive biotin and by Avidin blot. The K(m) for BCCP was approximately 5.2 microM and approximately 420 nM for biotin. MtBPL has low affinity (K(b) = 1.06x10(-6) M) for biotin relative to EcBirA but their K(m) are almost comparable suggesting that while the major function of MtBPL is biotinylation of BCCP, tight binding of biotin/bio-5'AMP by EcBirA is channeled for its repressor activity.

Conclusions/significance: These studies thus open up avenues for understanding the unique features of MtBPL and the role it plays in biotin utilization in M. tuberculosis.

Show MeSH
Related in: MedlinePlus