| Biotin synthase is a member of the adenosyl-methionine-dependent radical enzyme superfamily that requires electrons from flavodoxin to perform organic radical chemistry. Biotin synthase's target is dethiobiotin which needs its saturated carbons activated to facilitate sulfur insertion and generate the product biotin. The mechanism of this sulfur reaction has been hard to study, since there may yet be unknown components of the system, and in all studies to date the enzyme is limited to one turnover. The solution of the crystal structure of E. coli biotin synthase has brought light to a number of these questions regarding the sulfur donor and possible catalytic mechanisms. We have used the structure as a basis for examining protein residues on the surface which may be important in designating protein-protein specificity with flavodoxin. Interactions between the electron donor flavodoxin and biotin synthase have been determined through chemical crosslinking and mass spectrometry of the resulting covalent peptides. These results indicate that residues Lys58 and Lys226 are involved in a specific crosslink with acidic residues of flavodoxin. Mutagenesis of residues Lys49, Lys58, and Lys103 in the vicinity of the biotin synthase active site decreases its interaction with flavodoxin with a Kd 5- to 10-fold weaker than wild type (K d ∼ 1.3 muM). Hydrophobic residues within this patch of positive electrostatic surface potential on biotin synthase also help contribute to the specificity for flavodoxin. Based upon these results, we propose the loops containing the redox active cluster to be the binding interface on biotin synthase which would be the site for flavodoxin docking. |
