Crystal structure of D‐ribose‐5‐phosphate isomerase (RpiA) from Escherichia coli

Introduction. Ribose phosphate isomerase (D-ribose-5phosphate ketol-isomerase; EC 5.3.1.6; RPI), an enzyme involved in the first step of the non-oxidative branch of the pentose phosphate pathway, catalyzes the reversible conversion of ribose-5-phosphate to ribulose 5-phosphate. Two ribose phosphate isomerases, RpiA and RpiB, have been identified in Escherichia coli . RpiA, which is constitutively expressed, accounts for about 99% of the total RPI activity for strains grown in nutrient broth. E. coli strains defective in the rpiA gene are still able to use ribose as a carbon source due to the presence of the second RPI, a ribose-inducible RpiB. In contrast, the double mutant rpiA : rpiB shows severe inhibition of growth due to the accumulation of ribose-5-phosphate. RpiA, from E. coli, exists as a homodimer in solution with a subunit molecular weight of 22.8 kDa. Despite the differences in the amino acid sequence and molecular weight, both RpiA and RpiB can efficiently catalyze the interconversion of ribose-5phosphate and ribulose-5-phosphate. Sequence alignment of RPIs from various organisms shows a conservation of sequence around two regions (corresponding to RpiA)O[37] GXG(T/S)GST [43]O andO[81] DGADE-(X)8KGXG [97]O throughout the family. In RPIs, the transfer of protons between the first and the second carbon atoms of the pentose-5 -phosphate substrate has been suggested to proceed through a single base mechanism involving a cis-enediol(ate) intermediate. Through site-directed mutagenesis, Asp87 of spinach RPI has been suggested to play the role of a general base in the interconversion of ribose-5-phosphate and ribulose-5-phosphate. A similar acid-base mechanism has been suggested to function in rabbit phosphoglucose isomerase and triosephosphate isomerase from Leishmania. Although sequence information is available for RPIs from various organisms, no structural information for this family of enzyme is available. To understand more about this family of proteins, we have determined the crystal structure of E. coli RpiA at 2.5 Å resolution. Materials and methods. The gene encoding E. coli RpiA was cloned and the protein, labeled with selenomethionine, was expressed and purified as a N-terminal GST fusion. The GST tag was removed by thrombin cleavage before crystallization trials. Two crystal forms were obtained by the hanging drop vapor diffusion by equilibrating drops containing 2 L of protein (4.7 mg/mL) in buffer [50 mM Tris, pH 8.5, 400 mM NaCl, and 5 % (v/v) glycerol] and 2 L reservoir solution consisting of either (a) 1.05 M trisodium citrate, 0.1 M HEPES buffer, pH 7.5 or (b) 1.2 M ammonium sulfate, 3% (v/v) isopropanol, respectively, suspended over 1 mL of reservoir solution. Crystals of the former condition belong to space group P1 with unit cell dimensions a 42.6, b 42.6, c 60.8 Å, 101.3°, 90.5°, and 98.8° (Z 2), whereas the latter belong to orthorhombic space group C2221 with unit cell dimensions a 70.3 Å, b 71.8 Å, c 193.1 Å (Z 16). Further investigations were conducted by using the orthorhombic crystals. Before data collection, the crystals were soaked for 1 min in a cryoprotecting solution of 1.4 M ammonium sulfate, 3% (v/v) isopropanol, and 25% (v/v) glycerol and flash cooled to 100 K in a cold stream of N2 gas. The structure of RpiA was determined by MAD phasing from a SeMet-labeled protein crystal (Table I). Data processing and scaling were performed with HKL2000. Twelve of the expected 14 Se sites in the asymmetric unit were found with the program SOLVE by using 2.5 Å resolution data-yielding phases with an overall figure of merit (FOM) of 0.42. Density modification with the program RESOLVE improved the quality of the map and the overall FOM increased to 0.60. Approximately 70% of the protein main-chain and 45% of the side-chain atoms were built automatically with RESOLVE. Further model building was performed by using program O. The complete model was refined against the high-energy remote wavelength data set using program CNS version 1.0. Noncrystallographic symmetry restraints were applied throughout the refinement. During refinement, 10% of the reflections were set aside to monitor Rfree. Water mol-