Structure-Function Studies of Escherichia coli Biotin Synthase via a Chemical Modification and Site-Directed Mutagenesis Approach

Abstract

In Escherichia coli, biotin synthase (bioB gene product) catalyzes the key step in the biotin biosynthetic pathway, converting dethiobiotin (DTB) to biotin. Previous studies have demonstrated that BioB is a homodimer and that each monomer contains an iron-sulfur cluster. The purified BioB protein, however, does not catalyze the formation of biotin in a conventional fashion. The sulfur atom in the iron-sulfur cluster or from the cysteine residues in BioB have been suggested to act as the sulfur donor to form the biotin molecule, and yet unidentified factors were also proposed to be required to regenerate the active enzyme. In order to understand the catalytic mechanism of BioB, we employed an approach involving chemical modification and site-directed mutagenesis. The properties of the modified and mutated BioB species were examined, including DTB binding capability, biotin converting activity, and Fe(2+) content. From our studies, four cysteine residues (Cys 53, 57, 60, and 97) were assigned as the ligands of the iron-sulfur cluster, and Cys to Ala mutations completely abolished biotin formation activity. Two other cysteine residues (Cys 128 and 188) were found to be involved mainly in DTB binding. The tryptophan and histidine residues were suggested to be involved in DTB binding and dimer formation, respectively. The present study also reveals that the iron-sulfur cluster with its ligands are the key components in the formation of the DTB binding site. Based on the current results, a refined model for the reaction mechanism of biotin synthase is proposed.

Biological Radical Sulfur Insertion Reactions

“…However, when the enzyme returns to reductive anaerobic conditions, iron and sulfide are mobilized to regenerate a (4Fe−4S) cluster. − ,, The active enzyme form is the one containing the (4Fe−4S) cluster. Site-directed mutants of BioB in which the cysteines of the conserved sequence (Cys53, Cys57, Cys60) have been changed into alanine are inactive. − It has been reported that under certain reconstitution conditions each polypeptide can carry two clusters, one (4Fe−4S) cluster chelated by the cysteine triad and a (2Fe−2S) cluster presumably bound to three other conserved cysteines, Cys97, Cys128, and Cys188. − The presence of two different clusters in different sites in this type of enzyme preparation has been recently demonstrated by Mössbauer spectroscopy using differently substituted 57 Fe protein . The cluster composition of BioB is, however, not yet firmly established.…”

“…52,53 It is a homodimer of 77 kDa, with each polypeptide chain carrying an oxygensensitive (4Fe-4S) cluster, probably ligated by three cysteines of a CXXXCXXC box conserved among all BioB known sequences and a fourth still not identi- fied ligand. [54][55][56][57][58][59][60] In the presence of oxygen, the iron center is unstable and loses iron and sulfide, being converted into a (2Fe-2S) cluster. However, when the enzyme returns to reductive anaerobic conditions, iron and sulfide are mobilized to regenerate a (4Fe-4S) cluster.…”

“…Site-directed mutants of BioB in which the cysteines of the conserved sequence (Cys53, Cys57, Cys60) have been changed into alanine are inactive. [57][58][59][60] It has been reported that under certain reconstitution conditions each polypeptide can carry two clusters, one (4Fe-4S) cluster chelated by the cysteine triad and a (2Fe-2S) cluster presumably bound to three other con-served cysteines, Cys97, Cys128, and Cys188. [63][64][65] The presence of two different clusters in different sites in this type of enzyme preparation has been recently demonstrated by Mo ¨ssbauer spectroscopy using differently substituted 57 Fe protein.…”

“…The reaction involves activation/cleavage of C−H bonds of dethiobiotin and insertion of a sulfur atom (Figure ). The enzyme from E. coli , the product of the bioB gene, has been the most extensively studied biotin synthase and is named BioB in the following. , It is a homodimer of 77 kDa, with each polypeptide chain carrying an oxygen-sensitive (4Fe−4S) cluster, probably ligated by three cysteines of a CXXXCXXC box conserved among all BioB known sequences and a fourth still not identified ligand. − In the presence of oxygen, the iron center is unstable and loses iron and sulfide, being converted into a (2Fe−2S) cluster. However, when the enzyme returns to reductive anaerobic conditions, iron and sulfide are mobilized to regenerate a (4Fe−4S) cluster. − ,, The active enzyme form is the one containing the (4Fe−4S) cluster.…”

Biosynthesis of 2-Hydroxyethylphosphonate, an Unexpected Intermediate Common to Multiple Phosphonate Biosynthetic Pathways

“…Metal Identification-1,10-Phenanthroline was used to determine the iron content of a protein (20,21 and has a maximal absorbance at 510 nm. Each of the ADHs (3-5 M final concentration) was mixed with 1,10-phenanthroline (1.5-2 mM final concentration) and incubated at 4°C until the activity was completely abolished (usually 1-2 h).…”