Abstract
Dps (DNA protection during starvation) enzymes are a major class of dodecameric proteins that bacteria use to detoxify their cytosol through the uptake of reactive iron species. In the stationary growth phase of bacteria, Dps enzymes are primarily used to protect DNA by biocrystallization. To characterize the wild type Dps protein from Microbacterium arborescens that displays additional catalytic functions (amide hydrolysis and synthesis), we determined the crystal structure to a resolution of 2.05 Å at low iron content. The structure shows a single iron at the ferroxidase center coordinated by an oxo atom, one water molecule, and three ligating residues. An iron-enriched protein structure was obtained at 2 Å and shows the stepwise uptake of two hexahydrated iron atoms moving along channels at the 3-fold axis before a restriction site inside the channels requires removal of the hydration sphere. Supporting biochemical data provide insight into the regulation of this acylamino acid hydrolase. Moreover, the peroxidase activity of the protein was determined. The influence of iron and siderophores on the expression of acylamino acid hydrolase was monitored during several stages of cell growth. Altogether our data provide an interesting view of an unusual Dps-like enzyme evolutionarily located apart from the large Dps sequence clusters.
Highlights
Bacteria regulate iron homeostasis using iron-sensing repressors such as Fur in Escherichia coli [1,2,3]
Protein sequences related to Dps-like amino acid hydrolase (AAH) proteins were identified and submitted to clustering according to their similarity using the pairwise p values as artificial attractive forces
Most of these groups contain a large number of similar sequences (e.g. Actinobacteria, ␥-Proteobacteria, and Bacilli), whereas the closely AAH-related proteins form a smaller subcluster of only seven protein sequences
Summary
Western Blot Analysis—M. arborescens strain Se14 was cultivated in BHI medium (Roth). Initial crystals from the full screen diffracting to 3.5 Å were obtained in space group P212121 with four dodecamers of the enzyme in the asymmetric unit These crystals were further refined using the additive screen from Hampton Research at 18 °C by the hanging drop vapor diffusion method against 0.5 ml of the reservoir solution using chemicals that were all from Fluka. Crystal drops were prepared by mixing 1 l of protein at 11 mg/ml concentration (in 20 mM Tris-HCl, 50 mM NaCl (pH 8)) with 1 l of reservoir solution and 0.3 l of additive. Another crystal form was thereby obtained under the same conditions after 30 days using additives such as spermine, maltose, etc.
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