Eiji Inagaki

Cloning and Crystal Structure of Hematopoietic Prostaglandin D Synthase

Hematopoietic prostaglandin (PG) D synthase is the key enzyme for production of the D and J series of prostanoids in the immune system and mast cells. We isolated a cDNA for the rat enzyme, crystallized the recombinant enzyme, and determined the three-dimensional structure of the enzyme complexed with glutathione at 2.3 A resolution. The enzyme is the first member of the sigma class glutathione S-transferase (GST) from vertebrates and possesses a prominent cleft as the active site, which is never seen among other members of the GST family. The unique 3-D architecture of the cleft leads to the putative substrate binding mode and its catalytic mechanism, responsible for the specific isomerization from PGH2 to PGD2.

Structure of the Thermus thermophilus putative periplasmic glutamate/glutamine-binding protein

As part of a structural genomics project, the crystal structure of a 314-amino-acid protein encoded by Thermus thermophilus HB8 gene TT1099 was solved to 1.75 A using the multiple-wavelength anomalous dispersion (MAD) method and a selenomethionine-incorporated protein. The native protein structure was solved to 1.5 A using the molecular-replacement method. Both structures revealed a bound ligand, L-glutamate or L-glutamine, and a fold related to the periplasmic substrate-binding proteins (PSBP). Further comparative structural analysis with other PSBP-fold proteins revealed the conservation of the predicted membrane permease binding surface area and indicated that the T. thermophilus HB8 molecule is most likely to be an L-glutamate and/or an L-glutamine-binding protein related to the cluster 3 periplasmic receptors. However, the geometry of ligand binding is unique to the T. thermophilus HB8 molecule.

Structure of PIN-domain protein PH0500 from Pyrococcus horikoshii.

The Pyrococcus horikoshii OT3 protein PH0500 is highly conserved within the Pyrococcus genus of hyperthermophilic archaea and shows low amino-acid sequence similarity with a family of PIN-domain proteins. The protein has been expressed, purified and crystallized in two crystal forms: PH0500-I and PH0500-II. The structure was determined at 2.0 A by the multiple anomalous dispersion method using a selenomethionyl derivative of crystal form PH0500-I (PH0500-I-Se). The structure of PH0500-I has been refined at 1.75 A resolution to an R factor of 20.9% and the structure of PH0500-II has been refined at 2.0 A resolution to an R factor of 23.4%. In both crystal forms as well as in solution the molecule appears to be a dimer. Searches of the databases for protein-fold similarities confirmed that the PH0500 protein is a PIN-domain protein with possible exonuclease activity and involvement in DNA or RNA editing.

Expression, purification, crystallization and preliminary X‐ray diffraction analysis of galactokinase from Pyrococcus horikoshii. Erratum

Galactokinase (EC 2.7.1.6) catalyzes the ATP-dependent phosphorylation of alpha-D-galactose to alpha-D-galactose-1-phosphate, in an additional metabolic branch of glycolysis. The apo-form crystal structure of the enzyme has not yet been elucidated. Crystals of galactokinase from Pyrococcus horikoshii were prepared in both the apo form and as a ternary complex with alpha-D-galactose and an ATP analogue. Diffraction data sets were collected to 1.24 A resolution for the apo form and to 1.7 A for the ternary complex form using synchrotron radiation. The apo-form crystals belong to space group C2, with unit-cell parameters a = 108.08, b = 38.91, c = 81.57 A, beta = 109.8 degrees. The ternary complex form was isomorphous with the apo form, except for the length of the a axis. The galactokinase activity of the enzyme was confirmed and the kinetic parameters at 323 K were determined.

Crystallization and avoiding the problem of hemihedral twinning in crystals of Δ1-pyrroline-5-carboxylate dehydrogenase from Thermus thermophilus

Delta1-Pyrroline-5-carboxylate dehydrogenase from Thermus thermophilus (TtP5CDh) has been crystallized in a citrate-bound form (TtP5CDh-cit). The crystals diffracted to well beyond 2 A resolution, but exhibited perfect or near-perfect hemihedral twinning. Variation of crystallization conditions resulted in the growth of larger untwinned crystals or crystals with significantly reduced twin content, all with similar unit-cell parameters. The soaking of TtP5CDh-cit crystals in citrate-free solution produced crystals of the apo form (TtP5CDh-apo). The TtP5CDh-apo crystals belong to space group R3, with unit-cell parameters a = b = 102.29, c = 279.28 A, and diffract to 1.08 A. Crystals soaked in solution with NAD+ (TtP5CDh-NAD), NADH (TtP5CDh-NADH) and glutamate (TtP5CDh-Glu) were also prepared and characterized.