Crystal structure of <i>E. coli</i> yddE protein reveals a striking homology with diaminopimelate epimerase

Abstract

As part of our structural genomics effort in solving crystal structures of Escherichia coli proteins of unknown function with widespread distribution among several Gram+ and Gram− bacteria,1-3 we selected the yddE open-reading frame (ORF) that encodes a hypothetical protein of 297 amino acid residues with a theoretical molecular weight of 32.3 kDa. In the Pfam database, YddE is annotated as a hypothetical protein probably involved in phenazine biosynthesis (family PhzF/PhzC; accession number: PF02567) and members of this family are also distantly related to diaminopimelate (DAP) epimerase.4 Phenazines are derived from the shikimic acid pathway, and one of the derived metabolites, phenazine-1-carboxylic acid (PCA), is a broad-spectrum antibiotic widely used by bacteria against fungal root pathogens and may also act as virulence factors.5 Here, we present the crystal structures of YddE from E. coli in the native and selenomethionine (Se-Met) forms arising from two different crystal forms and determined with use of the medium-scale throughput platform of the Marseilles Structural Genomics Program. The structure of Se-Met YddE was solved at 2.5 Å resolution using the single-wavelength anomalous dispersion (SAD) method and refined to 2.05 Å resolution using the data set from the remote energy. The native protein was solved by the molecular replacement method using the Se-Met YddE coordinates as a template and refined to 2 Å-resolution. The yddE ORF was amplified from the genome of E. coli strain K-12 by polymerase chain reaction (PCR), and subcloned into the expression plasmid pDEST17 (Gateway, Invitrogen); the resulting construct encodes a protein extended at the N-terminus by 15 amino acids and 6 histidines.2, 6 Expression was carried out using E. coli Tuner (DE3)pLysS cells grown in Luria–Bertani (LB) medium at 37°C. Bacterial cells were lysed by treatment with lysozyme and consequent freeze-thawing. Nickel–ion affinity and subsequent preparative gel filtration chromatographies were performed using an Äkta FPLC (Amersham Biosciences). Se-Met YddE was produced using the same bacterial strain grown in minimal M9 medium and supplemented, before induction, with Se-Met and amino acids known to inhibit methionine biosynthesis.7 The purified proteins were further characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption/ionization (MALDI-TOF) mass spectroscopy, circular dichroism (CD), and dynamic light scattering. Sitting drop vapor diffusion crystallization screens were set-up with a TECAN Genesis robot on deep-well microtiter plates containing 1.5 μL of protein solution (14 mg/mL) and equal amounts of 240 crystallization conditions provided by commercial kits: Stura Footprint and Structure Screens (Molecular Dimensions Ltd., Cambridge, UK), Wizard I and II (Emerald BioStructures, Baimbridge Island, WA). Several hits appeared with various conditions, most of them containing low-molecular-weight polyethylene glycol (PEG) as precipitant in the pH range 6–8. Refinement of these conditions by the hanging drop vapor diffusion method lead to well-diffracting crystals, grown from 20% PEG 600, 0.1 M imidazole-malate pH 6.5. Crystals for the Se-Met protein (6 mg/mL) were grown from 12% polyethylene glycol monomethyl ether (MPEG) 5 K, 0.2 M Na-acetate pH 5.0. Crystals of the native and the Se-Met protein belong to space group P21 with 2 and 4 molecules in the asymmetric unit, respectively. Cryosolution consisted of the mother liquor for native crystals and supplemented with 5–10% glycerol for the Se-Met YddE crystals. All data sets were collected at 100 K on flash-frozen crystals. A 3-wavelength MAD data set for Se-Met YddE was collected on beam line ID14-EH4, and a data set for the native protein was collected on beam line ID14-EH1 (ESRF, Grenoble). Data were indexed and integrated with DENZO,8 and were scaled and merged with SCALA.9 The structure of YddE was solved by SAD on the S