Characterization of the vitamin B-12 receptor in Salmonella typhimurium

Photoaffinity labeling was employed in the peptide mapping of the Salmonella typhimurium vitamin B-12 receptor BtuB. Adenosylcobalamin (coenzyme B-12) a natural substrate of BtuB, was derivatized using the N-hydroxysuccinimide ester of 4-azidosalicylic acid (NHS-ASA) crosslinker. NHS-ASA was reacted with adenosylcobalsimin and radiolabeled with 125l to yield the derivatized product [125l]ASA-adenosylcobalamin. The derivatized product was photoaffinity-labeled to the BtuB binding site. Limited proteolysis was then performed using the Staphylococcus aureus V8 protease. The proteolytic fragments of the radiolabeled BtuB were detected using fluorography. The results indicated that the derivatization product [125l]ASA-adenosylcobalamin was selectively bound to BtuB, and upon flashing the bound complex with light, [125l]ASA-adenosylcobalamin was covalently linked to the BtuB binding site. N-terminal sequencing of a proteolytic fragment revealed the binding site to be at the BtuB segment NH2-Ala355-Ala356-Ala357-Arg358-Sei359-Asp360-Asp361-Asn362- Ser363-Gln364-Phe365. Amino acid substitutions by site-directed mutagenesis were performed at the identified binding site. [14C8]Adenosylcobalamin binding measurements by these mutant BtuB proteins showed that Arg367, Arg437, Arg487, and Lys394 played a role in the binding of adenosylcobalamin.