title: The Crystal Structure of the C-Terminal Domain of the Salmonella enterica PduO Protein: An Old Fold with a New Heme-Binding Mode.
abstract: The two-domain protein PduO, involved in 1,2-propanediol utilization in the pathogenic Gram-negative bacterium Salmonella enterica is an ATP:Cob(I)alamin adenosyltransferase, but this is a function of the N-terminal domain alone. The role of its C-terminal domain (PduOC) is, however, unknown. In this study, comparative growth assays with a set of Salmonella mutant strains showed that this domain is necessary for effective in vivo catabolism of 1,2-propanediol. It was also shown that isolated, recombinantly-expressed PduOC binds heme in vivo. The structure of PduOC co-crystallized with heme was solved (1.9 Å resolution) showing an octameric assembly with four heme moieities. The four heme groups are highly solvent-exposed and the heme iron is hexa-coordinated with bis-His ligation by histidines from different monomers. Static light scattering confirmed the octameric assembly in solution, but a mutation of the heme-coordinating histidine caused dissociation into dimers. Isothermal titration calorimetry using the PduOC apoprotein showed strong heme binding (K d = 1.6 × 10(-7) M). Biochemical experiments showed that the absence of the C-terminal domain in PduO did not affect adenosyltransferase activity in vitro. The evidence suggests that PduOC:heme plays an important role in the set of cobalamin transformations required for effective catabolism of 1,2-propanediol. Salmonella PduO is one of the rare proteins which binds the redox-active metabolites heme and cobalamin, and the heme-binding mode of the C-terminal domain differs from that in other members of this protein family.
authors.affiliation: Applied Genetics of Microorganisms, Department of Biology/Chemistry, University of Osnabrück Osnabrück, Germany.
authors.forename: Darío
authors.initials: D
authors.lastname: Ortiz de Orué Lucana
authors.affiliation: Centre of Excellence in Biocrystallography, Department of Chemical and Pharmaceutical Sciences, University of Trieste Trieste, Italy.
authors.forename: Neal
authors.initials: N
authors.lastname: Hickey
authors.affiliation: Division of Microbiology, Department of Biology/Chemistry, University of Osnabrück Osnabrück, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Hensel
authors.affiliation: Department of Physics, University of Osnabrück Osnabrück, Germany.
authors.forename: Johann P
authors.initials: JP
authors.lastname: Klare
authors.affiliation: Centre of Excellence in Biocrystallography, Department of Chemical and Pharmaceutical Sciences, University of Trieste Trieste, Italy.
authors.forename: Silvano
authors.initials: S
authors.lastname: Geremia
authors.affiliation: Centre for Bioinformatics, University of Hamburg Hamburg, Germany.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Tiufiakova
authors.affiliation: Centre for Bioinformatics, University of Hamburg Hamburg, Germany.
authors.forename: Andrew E
authors.initials: AE
authors.lastname: Torda
keywords: Salmonella; cobalamin; heme binding; protein structure and evolution; redox co-factor