Ken Ichi Tomita

Three-Dimensional Structure of a DNA Repair Enzyme, 3-Methyladenine DNA Glycosylase II, from Escherichia coli

The three-dimensional structure of Escherichia coli 3-methyladenine DNA glycosylase II, which removes numerous alkylated bases from DNA, was solved at 2.3 A resolution. The enzyme consists of three domains: one alpha + beta fold domain with a similarity to one-half of the eukaryotic TATA box-binding protein, and two all alpha-helical domains similar to those of Escherichia coli endonuclease III with combined N-glycosylase/abasic lyase activity. Mutagenesis and model-building studies suggest that the active site is located in a cleft between the two helical domains and that the enzyme flips the target base out of the DNA duplex into the active-site cleft. The structure of the active site implies broad substrate specificity and simple N-glycosylase activity.

The stacking interactions in 7-methylguanine-tryptophan systems, a model study for the interaction between the 'cap' structure of mRNA and its binding protein

Stacking interactions were shown by spectroscopic and X-ray crystallographic studies to be formed between the tryptophan and the protonated 7-methylguanine derivatives. These interactions would be in part responsible for the specific interaction between the 5-terminal capped structure of mRNA and its binding protein.

Bisphenanthrene ethers from Bletilla striata

Abstract From tubers of Bletilla striata , bisphenanthrene ethers were isolated and their structures were elucidated from their spectral data and X-ray analysis.

Blespirol, a phenanthrene with a spirolactone ring from Bletilla striata

Abstract A novel phenanthrene derivative with a spirolactone ring, blespirol, has been isolated from tubers of Bletilla striata and its structure elucidated on the basis of spectroscopic data and X-ray analysis.

The crystal structure of a major metabolite of thyroid hormone: 3,3′,5′-triiodo-L-thyronine

Two independent conformations of the thyroinactive thyroid hormone metabolite, 3,3,5-triiodo-L-thyronine (rT3) were determined by X-ray diffraction methods. The conformations show significant difference in the lettering geometry when compared with those of the thyroactive thyroxine (T4) and 3,5,3-triiodo-L-thyronine (T3). The diphenyl ether conformation of the two conformers of rT3 is an anti-skewed one, in which the torsion angles, phi (C5-C4-O4-Cl) are 8 degrees and -6 degrees, and phi (C4-O4-Cl-O6) are 86 degrees and 87 degrees. This conformation is in contrast to a twist-skewed one of T4 and T3. The difference in the binding abilities between T4, T3 and rT3 to thyroxine binding carrier proteins in serum or to a nuclear receptor protein may be explained by the characteristic solid-state conformations of these metabolites.

Three-dimensional structure of a mutant ribonuclease T1 (Y45W) complexed with non-cognizable ribonucleotide, 2′AMP, and its comparison with a specific complex with 2′GMP

The crystal structure of a mutant ribonuclease T1 (Y45W) complexed with a non-cognizable ribonucleotide, 2AMP, has been determined and refined to an R-factor of 0.159 using X-ray diffraction data at 1.7 A resolution. A specific complex of the enzyme with 2GMP was also determined and refined to an R-factor of 0.173 at 1.9 A resolution. The adenine base of 2AMP was found at a base-binding site that is far apart from the guanine recognition site, where the guanine base of 2GMP binds. The binding of the adenine base is mediated by a single hydrogen bond and stacking interaction of the base with the imidazole ring of His92. The mode of stacking of the adenine base with His92 is similar to the stacking of the guanine base observed in complexes of ribonuclease T1 with guanylyl-2,5-guanosine, reported by Koepke et al., and two guanosine bases, reported by Lenz et al., and in the complex of barnase with d(GpC), reported by Baudet & Janin. These observations suggest that the site is non-specific for base binding. The phosphate group of 2AMP is tightly locked at the catalytic site with seven hydrogen bonds to the enzyme in a similar manner to that of 2GMP. In addition, two hydrogen bonds are formed between the sugar moiety of 2AMP and the enzyme. The 2AMP molecule adopts the anti conformation of the glycosidic bond and C-3-exo sugar pucker, whereas 2GMP is in the syn conformation with C-3-endo-C-2-exo pucker. The mutation enhances the binding of 2GMP with conformational changes of the sugar ring and displacement of the phosphate group towards the interior of the catalytic site from the corresponding position in the wild-type enzyme complex. Comparison of two crystal structures obtained provides a solution to the problem that non-cognizable nucleotides exhibit unexpectedly strong binding to the enzyme, compared with high specificity in nucleolytic activity. The results indicate that the discrimination of the guanine base from the other nucleotide bases at the guanine recognition site is more effective than that estimated from nucleotide-binding experiments so far.

X-Ray crystal structure of sarothralin, a novel antibiotic compound from Hypericum japonicum

A novel antibiotic compound, sarothralin, from Hypericum japonicum Thunb. (Sarothra japonica Thunb.) has been isolated and its stereostructure elucidated using spectroscopin and X-ray Crystallographic techniques.

Crystallization and preliminary X-ray investigation of non-specific complexes of a mutant ribonuclease T1 (Y45W) with 2′AMP and 2′UMP

We have succeeded in crystallizing complexes of a mutant ribonuclease T1 (Y45W) with the non-cognizable ribonucleotides 2AMP and 2UMP by macroscopic seeding of microcrystals of the mutant enzyme complexed with 2GMP, which is the cognizable nucleotide inhibitor. The mutant enzyme has a tryptophan residue instead of Tyr45 of the wild-type enzyme and thus this mutation enhances the binding of ribonucleotides to the enzyme. The space group is P212121 with unit cell dimensions a = 49.40 A, b = 46.71 A, c = 41.02 A for the complex with 2AMP and a = 48.97, b = 46.58 A, c = 40.97 A for the complex with 2UMP, both of which are poorly isomorphous to the mother crystals. Diffraction data for the complexes with 2AMP and 2UMP were collected on a diffractometer at 1.7 A and 2.4 A resolution, respectively. The present studies show that crystallization of non-specific complexes of other protein-ligand systems with the dissociation constants around 10(-3) M, or even larger, could be feasible by application of the seeding technique. A comparison of the crystal structures of the complexes with that with 2GMP may serve as a structural basis for the determination of differences between the specific and non-specific interactions of the enzyme.

The crystal and molecular structrue of 8-methyladenosine 3′-monophosphate dihydrate

8-Methyladenosine 3-monophosphate dihydrate was synthesized and crystallized in the monoclinic space group P21 with the unit cell dimensions: a = 9.095(2) A, b = 16.750(3) A, c = 5.405(2) A and beta = 97.61(3) degrees. The structure was determined by the application of the heavy atom method and refined to give a final R factor of 0.047. The pertinent conformations are as follows: the syn conformation about the glycosyl bond (chiCN = 216.8 degrees), the C(2)-endo sugar puckering with the displacement of 0.55 A; and the gauche-gauche conformation about the C(4)-C(5) bond capable of forming an intramolecular hydrogen bonding between N(3) of adenine base and O(5) of the hydroxymethylene group on the ribose. The molecule exists in the zwitterionic form with the N(1) of the adenine base protonated by a phosphate proton and is stabilized by three-dimensional networks of hydrogen bonding through the crystalline water molecules or directly between the adjacent nucleotide molecules; no base stacking was observed.

Crystallization and preliminary X-ray diffraction studies of 3-methyladenine-DNA glycosylase II from Escherichia coli

Single crystals of 3-methyladenine-DNA glycosylase II produced from the alkA gene of Escherichia coli have been obtained by the method of vapour diffusion with polyethylene glycol 6000 as precipitant at pH 8.5. The crystals belong to the monoclinic space group C2, with a = 58.6 A, b = 76.8 A, c = 62.2 A, beta = 110.3 degrees. They contain one molecule per asymmetric unit and diffract to 2.7 A resolution.