RESUMEN
The structure of a putative protease from Bacteroides thetaiotaomicron features an unprecedented binding site for flavin mononucleotide. The flavin isoalloxazine ring is sandwiched between two tryptophan residues in the interface of the dimeric protein. We characterized the recombinant protein with regard to its affinity for naturally occurring flavin derivatives and several chemically modified flavin analogs. Dissociation constants were determined by isothermal titration calorimetry. The protein has high affinity to naturally occurring flavin derivatives, such as riboflavin, FMN, and FAD, as well as lumichrome, a photodegradation product of flavins. Similarly, chemically modified flavin analogs showed high affinity to the protein in the nanomolar range. Replacement of the tryptophan by phenylalanine gave rise to much weaker binding, whereas in the tryptophan to alanine variant, flavin binding was abolished. We propose that the protein is an unspecific scavenger of flavin compounds and may serve as a storage protein in vivo.
Asunto(s)
Proteínas Bacterianas/química , Bacteroides/enzimología , Mononucleótido de Flavina/química , Flavina-Adenina Dinucleótido/química , Péptido Hidrolasas/química , Riboflavina/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteroides/genética , Mononucleótido de Flavina/genética , Mononucleótido de Flavina/metabolismo , Flavina-Adenina Dinucleótido/genética , Flavina-Adenina Dinucleótido/metabolismo , Genómica , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Riboflavina/genética , Riboflavina/metabolismoRESUMEN
The substrates for Bacillus subtilis PLP synthase (YaaD and YaaE) are identified, and the first reconstitution of PLP biosynthesis using this pathway is described. Three partial reactions catalyzed by YaaD are also identified.