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


RESULTS
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
These studies thus open up avenues for understanding the unique features of MtBPL and the role it plays in biotin utilization in M. tuberculosis.
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
Sudha
S
Purushothaman
Garima
G
Gupta
Richa
R
Srivastava
Vasanthakumar Ganga
VG
Ramu
Avadhesha
A
Surolia
D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D006860:Hydrogen Bonding; D008024:Ligands; D008025:Ligases / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013816:Thermodynamics
D017931:DNA Primers; D008024:Ligands; D008025:Ligases