RESUMEN

A recently described putative protease from the gut bacterium Bacteroides thetaiotaomicron (termed ppBat) exhibits two tryptophan residues in the interface which enable specific binding of the isoalloxazine heterocycle of riboflavin and its two cofactor forms, FMN and FAD. Recombinant ppBat was used to capture riboflavin from bovine milk directly without any prior preparation steps. The flavin-loaded protein was then re-isolated by means of affinity chromatography to identify and quantify the captured flavins. Free riboflavin concentrations were determined to 197 and 151µg/l for milk with 3.5% and 0.5% fat content, respectively. Total riboflavin concentrations were also determined after acid-treatment of milk and were 4-5 times higher than for free riboflavin. Free FMN and FAD were not detectable and only trace amounts of FMN were found in milk following acid treatment. The method appears to be amenable to develop a direct assay for free riboflavin in milk and other foods.

Asunto(s)

Proteínas de la Leche/química , Leche/química , Riboflavina/química , Animales , Bovinos , Unión Proteica

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/metabolismo