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.
Highlights
Salmonella enterica is a food-borne Gram-negative pathogen causing severe gastroenteritis and systemic disease in animals and humans, growing under both aerobic and anaerobic conditions (Haraga et al, 2008; Hayward et al, 2015)
The maximum-likelihood phylogram in Figure 2 gives the evolutionary context of this work
The labeling is sparse, but the density of the nodes shows the number of related sequences
Summary
Salmonella enterica is a food-borne Gram-negative pathogen causing severe gastroenteritis and systemic disease in animals and humans, growing under both aerobic and anaerobic conditions (Haraga et al, 2008; Hayward et al, 2015). A New Heme-Binding Protein Domain is metabolized within a micro-compartment formed by a protein shell (Chowdhury et al, 2014; Bobik et al, 2015) This would help concentrate reactants and enzymes, and protect the system from side-reactions. Amongst its set of proteins, the pdu operon encodes PduO This is an ATP:Cob(I)alamin adenosyltransferase (ACA) catalyzing the synthesis of adenosylcobalamin (AdoCbl; coenzyme B12). The Salmonella PduO is a two-domain protein, but only the N-terminal domain is necessary for the transferase activity (Johnson et al, 2004). This was made clear by the related protein from Lactobacillus reuteri (LrPduO). This C-terminal domain, and its structure and function are the focus of this work
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