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Attachment of biotin to acceptor proteins occurs in a two-step reaction. First, BirA binds biotin and ATP to synthesize Bio-5′-AMP (biotinoyl-5'-adenylate) with release of pyrophosphate. In the second step the conserved lysine residue of the acceptor protein acts as a nucleophile and attacks the mixed anhydride bond to give the biotinylated acceptor protein plus AMP.
