Function of a conserved sequence motif in biotin holoenzyme synthetases

The biotin holoenzyme synthetases (BHS) are essential enzymes in all organi sms that catalyze post-translational linkage of biotin to biotin-dependent carboxylases. The primary sequences of a large number of these enzymes are now available and homologies are found among all. The glycine-rich sequence , GRGRXG, constitutes one of the homologous regions in these enzymes and, b ased on its similarity to sequences found in a number of mononucleotide bin ding enzymes, has been proposed to function in ATP binding in the BHSs. In the Escherichia coli enzyme, the only member of the family for which a thre e-dimensional structure has been determined, the conserved sequence is foun d in a partially disordered surface loop. Mutations in the sequence have pr eviously been isolated and characterized in vivo. In this work these single -site mutants, G115S, R118G, and R119W, of the E. coli BHS have been purifi ed and biochemically characterized with respect to binding of small molecul e substrates and the intermediate in the biotinylation reaction. Results of this characterization indicate that, rather than functioning in ATP bindin g, this glycine-rich sequence is required for binding the substrate biotin and the intermediate in the biotinylation reaction, biotinyl-5'-AMP. These results are of general significance for understanding structure-function re lationships in biotin holoenzyme synthetases.