Abstract

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.

Highlights

  • The structure of a putative protease from Bacteroides thetaiotaomicron has a unique binding site for a flavin

  • In a recent survey of flavin-dependent proteins, we identified a putative protease from Bacteroides thetaiotaomicron, a microbe inhabiting the human gut [1, 2] that presumably binds FMN [3]

  • Expression and Purification of putative protease from B. thetaiotaomicron (ppBat)—Cells transformed with the plasmid carrying the gene of ppBat were cultured, and protein production was induced by isopropyl ␤-D-thiogalactopyranoside

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Summary

Background

The structure of a putative protease from Bacteroides thetaiotaomicron has a unique binding site for a flavin. We report the biochemical characterization of the flavin-binding site of the protein and demonstrate that the recombinant protein is capable of binding naturally occurring flavin derivatives, such as lumichrome, riboflavin, FMN, and FAD (supplemental Fig. S1A) and a variety of chemically modified “artificial” flavin analogs, such as iso-riboflavin (6.7-dimethyl-8-nor-riboflavin) and iso-FMN as well as 8-amino- and 1- and 5-deaza-riboflavin [12,13,14] (supplemental Fig. S1B). It appears that this putative protease from B. thetaiotaomicron (ppBat) unspecifically. We propose that ppBat may serve as a flavin storage protein in gut bacteria

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