The non-heme iron dioxygenase PtlH from your soil organism is a member of the iron(II)/-ketoglutarateCdependent dioxygenase superfamily and catalyzes an essential reaction in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone. cysteine residue (11). An inducible, pentalenolactone-insensitive GAPDH isozyme that is part of the biosynthetic gene cluster confers self-resistance against the antibiotic in and additional strains (1,12C14). Open in a separate window Number 1 The PtlH reaction (A) The sesquiterpenoid antibiotic pentalenolactone produced by (Genbank NP_824167, 311 amino acids) was amplified by PCR from DNA of cosmid clone CL_216_D07 and cloned into the pET28e vector (a variant of pET28a [Novagen] in which the gene by PCR from cosmid CL_216_D07 using the ahead primer (5-CGCG TCTCGCTCATATGACGAACGTGCTGGGGA CTAC-3) and the same reverse primer as that previously used for the unnatural 311-aa version of PtlH (5-GGCCGGAAGCTTACTAGTCAAT TGTCATTCCACGTCGGTGGGGGTA-3) and subcloned the DNA for the shorter protein sequence into the pET28e vector. The resultant recombinant protein was over-expressed in BL21(DE3) (Invitrogen) at 20 C using 0.4 mM IPTG. Bacterial cells were lysed by ultrasonification on snow. The soluble protein was bound to nickel-agarose affinity resin (Qiagen), washed having a buffer comprising 20 mM Tris (pH 8.5), 250 mM NaCl, and 10 mM imidazole. His6-tagged protein was then eluted having a buffer comprising 20 mM Tris (pH 8.5), 250 mM NaCl, and 150 mM imidazole. The protein was further purified with anion exchange chromatography at pH 8.5, using a linear gradient of 10 mM to 1 1 LP-533401 pontent inhibitor M NaCl concentration, and size exclusion chromatography at pH 8.5 and 200 mM NaCl. The purified protein was concentrated to 25 mg/ml inside a buffer comprising 10 mM Tris (pH 8.5), 20 mM NaCl, and 7% glycerol. The sample was flash-frozen in liquid nitrogen and stored at ?80 C. The N-terminal histidine-tag was eliminated by thrombin digestion before crystallization. For the production of selenomethionyl proteins, the expression construct was transformed into B834(DE3) cells (Novagen). The bacterial growth was carried out in defined LeMaster press (29), and the protein was purified using the same protocol as for the wild-type protein. Crystals of PtlH were acquired at 4C with the sitting drop vapor diffusion method. The reservoir answer contained 100 mM TRIS pH 8.5, 200 mM MgCl2 and 20% PEG 3350 (w/v). 0.8 l of protein solution (13 mg/ml, containing 2 mM -keto- glutarate and 20 mM DTT) were mixed with 0.8 l of reservoir solution. Initial small crystals grew within 24 hours and larger crystals were cultivated by microseeding techniques. Crystals grew to maximum sizes of 0.45 0.12 0.12 mm. The crystals were cryo-protected by quick soaking in a solution comprising mother liquor with the help of 20% LP-533401 pontent inhibitor (v/v) ethylene glycol and adobe flash freezing in liquid nitrogen. The PtlH/-ketoglutarate/=?BL21(DE3) under the same conditions utilized for the wild-type protein and the resultant in four crystal forms at a resolution of up to 1.31 ? (Table 1). Crystal form I was acquired at pH 8.5 in space group P21 like a complex with Fe(II), -ketoglutarate and the non-reactive substrate enantiomer em ent /em -1-deoxypentalenic acid ( em ent /em -1PL, Fig. 1c) after soaking crystal form II having a racemic substrate combination for one hour. Crystal form III crystallized in space group P21 like a complex with SLCO2A1 iron and the -ketoglutarate-analogue inhibitor N-oxalylglycine (OGA) upon decreasing the pH of the precipitant to 7.5. LP-533401 pontent inhibitor Crystal form IV crystallized at pH 7.5 in space group P21212 like a complex with iron, OGA and contained a partially occupied molecule of em ent /em -1PL after soaking for quarter-hour. All crystal forms needed the presence of either -ketoglutarate or oxalylglycine and no crystals could be acquired with succinate..