title: Structure and stability of an unusual zinc-binding protein from Bacteroides thetaiotaomicron.
abstract: The crystal structure of a putative protease from Bacteroides thetaiotaomicron (ppBat) suggested the presence of a zinc ion in each protomer of the dimer as well as a flavin in the dimer interface. Since the chemical identity of the flavin and the exact mode of binding remained unclear, we have determined the crystal structure of ppBat in complex with riboflavin. The obtained structure revealed that the isoalloxazine ring is sandwiched between two tryptophan residues (Trp164) from both chains and adopts two alternate orientations with the N(10)-ribityl side chain protruding from the binding site in opposite directions. In order to characterize the zinc-binding site, we generated two single variants and one double variant in which the two coordinating cysteine residues (Cys74 and Cys111) were replaced by alanine. All three variants were unable to bind zinc demonstrating that both cysteine residues are essential for binding. Moreover, the lack of zinc binding also resulted in drastically reduced thermal stability (11-15°C). A similar effect was obtained when wild-type protein was incubated with EDTA supporting the conclusion that the zinc-binding site plays an important structural role in ppBat. On the other hand, attempts to identify proteolytic activity failed suggesting that the zinc may not act as a catalytic center in ppBat. Structurally similar zinc binding motives in other proteins were also found to play a structural rather than catalytic role and hence it appears that neither the flavin nor the zinc binding sites possess a catalytic function in ppBat.
authors.affiliation: Institute of Biochemistry, Graz University of Technology, A-8010 Graz, Austria.
authors.forename: Tanja
authors.initials: T
authors.lastname: Knaus
authors.affiliation: Institute of Molecular Biosciences, University of Graz, A-8010 Graz, Austria.
authors.forename: Michael K
authors.initials: MK
authors.lastname: Uhl
authors.affiliation: Institute of Biochemistry, Graz University of Technology, A-8010 Graz, Austria.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Monschein
authors.affiliation: Institute of Biochemistry, Graz University of Technology, A-8010 Graz, Austria.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Moratti
authors.affiliation: Institute of Molecular Biosciences, University of Graz, A-8010 Graz, Austria.
authors.forename: Karl
authors.initials: K
authors.lastname: Gruber
authors.affiliation: Institute of Biochemistry, Graz University of Technology, A-8010 Graz, Austria. Electronic address: peter.macheroux@tugraz.at.
authors.forename: Peter
authors.initials: P
authors.lastname: Macheroux
keywords: Flavin; Microcalorimetry; Redox potential; Thermal stability; X-ray crystallography; Zinc