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 (Kd = 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.

DeepDrug3D: Classification of ligand-binding pockets in proteins with a convolutional neural network

“…The second example is the C-terminal domain of a protein PduO (PduOC) playing an important role in the catabolism of 1,2-propanediol in the pathogenic bacterium Salmonella enterica (PDB-ID: 5cx7, chain E) [24]. Fig 8C presents the structure of PduOC co-crystallized with heme, whose binding mode significantly differs from those in other members of this protein family.…”

“…For instance, heme was identified as a physiological ligand of orphan nuclear receptors REV-ERBα and REV-ERBβ, extending the collection of known ligands of the human nuclear receptor family beyond endocrine hormones and dietary lipids [23]. Despite the fact that the PDB contains about 3,600 proteins complexed with some form of heme, new binding modes of heme are still being discovered [24]. Therefore, a reliable classification and characterization of binding sites in hypothetical proteins may support studies focused on the identification of physiological ligands and their binding modes.…”

EfgA is a conserved formaldehyde sensor that halts bacterial translation in response to elevated formaldehyde

“…Proteins with DUF336 domains are present in single- or multi-domain proteins and their genetic association with gene clusters that encode well-characterized pathways such as glycolate utilization in E. coli (GlcG), 1,2-propanediol utilization in Salmonella enterica (PduO), and chloroaniline degradation in Diaphorobacter sp. PCA039 (OrfU2) has been noted [82–86]. The two best characterized examples are HbpS of S. reticuli and PduO of S. enterica .…”

“…HbpS is located extracellularly where it senses and degrades heme and activates a two-component system involved in oxidative stress [88,92,117,118]. PduO is localized to the pdu microcompartment its DUF336 domain is fused to an ATP:cob(I)alamin adenosyltransferase domain; its DUF336 domain is not required for the activity of the ATP:Cob(I)alamin adenosyltransferase domain in vitro but is required for optimal 1,2-propanediol utilization for unknown reasons [86]. Both HbpS and PduO have been shown to bind heme and cobalamin but their cellular locations are distinct, consistent with their disparate functions.…”

A New Class of EutT ATP:Co(I)rrinoid Adenosyltransferases Found in Listeria monocytogenes and Other Firmicutes Does Not Require a Metal Ion for Activity

“…AdoCbl is the coenzyme of the 1,2-propanediol dehydratase that converts 1,2-propanediol to propionaldehyde 13 . In-depth studies of CobA and PduO have been reported, and structures with substrates bound to them have been elucidated 6, 14–16 . EutT class of ACATs are not as well understood.…”