Mitsuaki Sugahara

Crystal structure of a repair enzyme of oxidatively damaged DNA. MutM (FPG), from an extreme thermophile, Thermus thermophilus HB8

The MutM [formamidopyrimidine DNA glycosylase (Fpg)] protein is a trifunctional DNA base excision repair enzyme that removes a wide range of oxidatively damaged bases (N‐glycosylase activity) and cleaves both the 3′‐ and 5′‐phosphodiester bonds of the resulting apurinic/apyrimidinic site (AP lyase activity). The crystal structure of MutM from an extreme thermophile, Thermus thermophilus HB8, was determined at 1.9 Å resolution with multiwavelength anomalous diffraction phasing using the intrinsic Zn2+ ion of the zinc finger. MutM is composed of two distinct and novel domains connected by a flexible hinge. There is a large, electrostatically positive cleft lined by highly conserved residues between the domains. On the basis of the three‐dimensional structure and taking account of previous biochemical experiments, we propose a DNA‐binding mode and reaction mechanism for MutM. The locations of the putative catalytic residues and the two DNA‐binding motifs (the zinc finger and the helix–two‐turns–helix motifs) suggest that the oxidized base is flipped out from double‐stranded DNA in the binding mode and excised by a catalytic mechanism similar to that of bifunctional base excision repair enzymes.

Structural basis of leukotriene B4 12-hydroxydehydrogenase/15-oxo-prostaglandin 13-reductase catalytic mechanism and a possible SH3 binding loop

The bifunctional leukotriene B4 12-hydroxydehydrogenase/15-oxo-prostaglandin 13-reductase (LTB4 12-HD/PGR) is an essential enzyme for eicosanoid inactivation. It is involved in the metabolism of the E and F series of 15-oxo-prostaglandins (15-oxo-PGs), leukotriene B4 (LTB4), and 15-oxo-lipoxin A4 (15-oxo-LXA4). Some nonsteroidal anti-inflammatory drugs (NSAIDs), which primarily act as cyclooxygenase inhibitors also inhibit LTB4 12-HD/PGR activity. Here we report the crystal structure of the LTB4 12-HD/PGR, the binary complex structure with NADP+, and the ternary complex structure with NADP+ and 15-oxo-PGE2. In the ternary complex, both in the crystalline form and in solution, the enolate anion intermediate accumulates as a brown chromophore. PGE2 contains two chains, but only the ω-chain of 15-oxo-PGE2 was defined in the electron density map in the ternary complex structure. The ω-chain was identified at the hydrophobic pore on the dimer interface. The structure showed that the 15-oxo group forms hydrogen bonds with the 2′-hydroxyl group of nicotine amide ribose of NADP+ and a bound water molecule to stabilize the enolate intermediate during the reductase reaction. The electron-deficient C13 atom of the conjugated enolate may be directly attacked by a hydride from the NADPH nicotine amide in a stereospecific manner. The moderate recognition of 15-oxo-PGE2 is consistent with a broad substrate specificity of LTB4 12-HD/PGR. The structure also implies that a Src homology domain 3 may interact with the left-handed proline-rich helix at the dimer interface and regulate LTB4 12-HD/PGR activity by disruption of the substrate binding pore to accommodate the ω-chain.

Structure of aldolase from Thermus thermophilus HB8 showing the contribution of oligomeric state to thermostability.

2-Deoxyribose-5-phosphate aldolase catalyzes a reversible aldol condensation of two aldehydes via formation of a covalent Schiff-base intermediate at the active lysine residue. The crystal structure of 2-deoxyribose-5-phosphate aldolase from Thermus thermophilus HB8 has been determined with and without the substrate at atomic resolution. This enzyme, which has a unique homotetramer structure, has been compared with the previously reported crystal structures of two orthologues from Escherichia coli and Aeropyrum pernix. In contrast to the similar alpha/beta-barrel fold of the monomers, substantial quaternary structural differences are observed between these three enzymes. Further comparison of the subunit-subunit interface areas of these aldolases showed a clear positive correlation between the interface area and the living temperature of the source organism. From these results, it is concluded that the oligomeric state of 2-deoxyribose-5-phosphate aldolase is important for the thermostability and not for the catalytic function.

Crystallization screening test for the whole-cell project on Thermus thermophilus HB8.

It was essential for the structural genomics of Thermus thermophilus HB8 to efficiently crystallize a number of proteins. To this end, three conventional robots, an HTS-80 (sitting-drop vapour diffusion), a Crystal Finder (hanging-drop vapour diffusion) and a TERA (modified microbatch) robot, were subjected to a crystallization condition screening test involving 18 proteins from T. thermophilus HB8. In addition, a TOPAZ (microfluidic free-interface diffusion) designed specifically for initial screening was also briefly examined. The number of diffraction-quality crystals and the time of appearance of crystals increased in the order HTS-80, Crystal Finder, TERA. With the HTS-80 and Crystal Finder, the time of appearance was short and the rate of salt crystallization was low. With the TERA, the number of diffraction-quality crystals was high, while the time of appearance was long and the rate of salt crystallization was relatively high. For the protein samples exhibiting low crystallization success rates, there were few crystallization conditions that were common to the robots used. In some cases, the success rate depended greatly on the robot used. The TOPAZ showed the shortest time of appearance and the highest success rate, although the crystals obtained were too small for diffraction studies. These results showed that the combined use of different robots significantly increases the chance of obtaining crystals, especially for proteins exhibiting low crystallization success rates. The structures of 360 of 944 purified proteins have been successfully determined through the combined use of an HTS-80 and a TERA.

Crystal structure of dehydroquinate synthase from Thermus thermophilus HB8 showing functional importance of the dimeric state.

Introduction. Dehydroquinate synthase (DHQS) is a -nicotinamide adenine dinucleotide (NAD )-dependent metalloenzyme that converts 3-deoxy-D-arabino-heptulosonate-7-phosphate to dehydroquinate in the shikimate pathway of bacteria, microbial eukaryotes, and plants. This enzyme is expected to be one of the targets for novel antifungal and antibacterial drug designs, because the shikimate pathway is absent in mammals, and DHQS is required for pathogenic virulence. DHQS is known to have a multistep mechanism, where a single active site catalyzes 5 sequential reactions involving alcohol oxidation, phosphate elimination, carbonyl reduction, ring opening, and intramolecular aldol condensation. The crystal structure of DHQS from Aspergillus nidulans (AnDHQS) in complex with substrates and metal ions elucidated important residues in the active site and ligand-induced intersubunit orientational changes that provide a potential reaction mechanism. The AnDHQS structures also revealed their dimeric state in crystal form, although its biological significance was not fully discussed. This time, we have solved the crystal structure of DHQS from Thermus thermophilus HB8 (TtDHQS) at 1.8 Å resolution, which is the first crystal structure of this enzyme from thermophilic organisms. We show that TtDHQS is present as a homodimer in crystal form, as well as in solution. In this article, we present the structural comparison between AnDHQS and TtDHQS, providing insight into the biological significance of the dimeric state of this enzyme.

Evaluation of crystalline objects in crystallizing protein droplets based on line-segment information in greyscale images

Several automated crystallization systems have recently been developed for high-throughput X-ray structure analysis. However, the evaluation process for the growth state of crystallizing protein droplets has not yet been completely automated. This paper proposes a new evaluation method for crystalline objects in automated crystallization experiments. The main objective is to determine whether a droplet contains crystals suitable for diffraction experiments and analysis. The evaluation method developed here involves extracting line-segment features from an image of the droplet and discriminating the state of crystallization using classifiers based on line features. In order to verify the efficacy of the proposed method, it was used to classify images obtained by an automated crystallization system.

Evaluation of protein crystallization states based on texture information derived from greyscale images

In recent years, several projects have advanced research and development related to the automation of the protein crystallization process. However, evaluation of crystallization states has not yet been completely automated. In the usual crystallization process, researchers evaluate the protein crystallization growth states based on visual impressions and assign them a score over and over again. The method presented here automates this evaluation process. This method attempts to categorize the individual crystallization droplet images into five classes. The algorithm is comprised of pre-processing, feature extraction from images using texture analysis and a categorization process using linear discriminant analysis. The performance of this method has been evaluated by comparing the results obtained by using this method with the results from a human expert and the concordance rate was 90.6%.

Crystallization and preliminary X-ray diffraction studies of a DNA excision repair enzyme, UvrB, from Thermus thermophilus HB8

A DNA excision repair enzyme, UvrB, from Thermus thermophilus HB8 was crystallized by the vapor-diffusion method using lithium sulfate as the precipitant and beta-octylglucoside as an additive. The crystals belong to the trigonal space group P3121 or P3221, with unit-cell dimensions of a = b = 136.0 and c = 108.1 A. The crystal is most likely to contain one UvrB protein in an asymmetric unit with the Vm value of 3.8 A3 Da-1. The crystals diffracted X-rays beyond 2.9 A resolution. Although the crystals were sensitive to X-ray irradiation at room temperature, the frozen crystals at 100 K showed no apparent decay during the intensity measurement.

Structure of peptidyl-tRNA hydrolase 2 from Pyrococcus horikoshii OT3: insight into the functional role of its dimeric state.

Peptidyl-tRNA hydrolases catalyze the hydrolytic removal of the peptidyl moiety from the peptidyl-tRNA molecule to prevent misreading during translation. Here, the expression, purification, crystallization and X-ray diffraction study of peptidyl-tRNA hydrolase 2 from Pyrococcus horikoshii OT3 (PhPth2) are described. The crystal structures were determined as similar biological dimers in two different forms: P4(1)2(1)2 at 1.2 A resolution (form 1) and P4(3)22 at 1.9 A resolution (form 2). In the form 1 structure, the asymmetric unit contains one PhPth2 subunit and a crystallographic twofold axis defines the dimeric association with the cognate subunit. In the form 2 structure, there are two PhPth2 subunits in the asymmetric unit that make a similar dimer with a noncrystallographic twofold axis. In order to evaluate the thermodynamic stability, the intra-protomer and inter-protomer interactions of PhPth2 were analyzed and compared with those of other Pth2-family members. The thermodynamic parameters show that the large number of ion pairs compared with family members from other mesophilic organisms would contribute to the thermostability of PhPth2. The structural difference between the two dimers was quantitatively evaluated by a multiple C(alpha)-atom superposition. A significant structural difference between the two dimers was observed around the putative active site of this enzyme. A rigid-body rotation takes place so as to retain the dimeric twofold symmetry, suggesting positive cooperativity upon tRNA binding. The mechanism of ligand binding was further investigated using a docking model with a tRNA molecule. The docking study suggests that the binding of tRNA requires its simultaneous interaction with both subunits of the PhPth2 dimer.

Evaluation of protein crystallization state by sequential image classification

Purpose – The purpose of this paper is to present classification schemes for the crystallization state of proteins utilizing image processing.Design/methodology/approach – Two classification schemes shown here are combined sequentially.Findings – The correct ratio of experimental result using the method presented here is approximately 70 per cent.Originality/value – The paper is a contribution to automated evaluation crystal growth, combining two classifiers based on specific visual feature, sequentially.