Nobuhiro Mizuno

X-ray Crystallographic Analysis of 6-Aminohexanoate-Dimer Hydrolase MOLECULAR BASIS FOR THE BIRTH OF A NYLON OLIGOMER-DEGRADING ENZYME

6-Aminohexanoate-dimer hydrolase (EII), responsible for the degradation of nylon-6 industry by-products, and its analogous enzyme (EII′) that has only ∼0.5% of the specific activity toward the 6-aminohexanoate-linear dimer, are encoded on plasmid pOAD2 of Arthrobacter sp. (formerly Flavobacterium sp.) KI72. Here, we report the three-dimensional structure of Hyb-24 (a hybrid between the EII and EII′ proteins; EII′-level activity) by x-ray crystallography at 1.8 Å resolution and refined to an R-factor and R-free of 18.5 and 20.3%, respectively. The fold adopted by the 392-amino acid polypeptide generated a two-domain structure that is similar to the folds of the penicillin-recognizing family of serine-reactive hydrolases, especially to those of d-alanyl-d-alanine-carboxypeptidase from Streptomyces and carboxylesterase from Burkholderia. Enzyme assay using purified enzymes revealed that EII and Hyb-24 possess hydrolytic activity for carboxyl esters with short acyl chains but no detectable activity for d-alanyl-d-alanine. In addition, on the basis of the spatial location and role of amino acid residues constituting the active sites of the nylon oligomer hydrolase, carboxylesterase, d-alanyl-d-alanine-peptidase, and β-lactamases, we conclude that the nylon oligomer hydrolase utilizes nucleophilic Ser112 as a common active site both for nylon oligomer-hydrolytic and esterolytic activities. However, it requires at least two additional amino acid residues (Asp181 and Asn266) specific for nylon oligomer-hydrolytic activity. Here, we propose that amino acid replacements in the catalytic cleft of a preexisting esterase with the β-lactamase fold resulted in the evolution of the nylon oligomer hydrolase.

Crystal Structure of Dissimilatory Sulfite Reductase D (DsrD) Protein-Possible Interaction with B- and Z-DNA by Its Winged-Helix Motif

The crystal structure of DsrD from Desulfovibrio vulgaris Hildenborough has been determined at 1.2 A resolution. DsrD is in a dimeric form in the crystal, and five sulfate anions were located on the surface. The structure of DsrD comprises a winged-helix motif, which shows the highest structural homology to similar motifs found in Z-DNA binding proteins and some B-DNA binding proteins. The core structure of the molecule is constructed by intramolecular interactions of hydrophobic residues, which are well conserved in DNA binding proteins, suggesting that these proteins belong to the same superfamily on the basis of the structure. These results indicate a possible role of DsrD in transcription or translation of genes for enzymes catalyzing dissimilatory sulfite reduction.

Crystallization and preliminary neutron analysis of the dissimilatory sulfite reductase D (DsrD) protein from the sulfate-reducing bacterium Desulfovibrio vulgaris

Dissimilatory sulfite reductase D (DsrD) from Desulfovibrio vulgaris has been crystallized for a neutron diffraction study. The initial crystals obtained were too small for the neutron experiment. In order to obtain a larger crystal (>1 mm3), a combination of two techniques was developed to determine the optimum crystallization conditions: a crystallization phase diagram was obtained, followed by crystal-quality assessment via X-ray diffraction. Using conditions determined in this manner, a large single crystal (1.7 mm3) of DsrD protein was subsequently grown in D(2)O solution by the macroseeding technique. A neutron diffraction experiment was carried out using the BIX-3 diffractometer at the Japan Atomic Energy Research Institute (JAERI), collecting data to 2.4 A resolution from an optimized crystal.

Crystallization and preliminary X-ray crystallographic studies of the axin DIX domain

Axin is a negative regulator of the canonical Wnt signalling pathway that mediates the phosphorylation of beta-catenin by glycogen synthase kinase 3beta. The DIX domain of rat axin, which is important for its homooligomerization and interactions with other regulators in the Wnt pathway, was purified and crystallized by the sitting-drop vapour-diffusion technique using polyethylene glycol 6000 and lithium sulfate as crystallization agents. Crystals belong to space group P6(1) or P6(5), with unit-cell parameters a = b = 91.49, c = 84.92 A. An X-ray diffraction data set has been collected to a nominal resolution of 2.9 A.

Remote access and automation of SPring-8 MX beamlines

At SPring-8 MX beamlines, a remote access system has been developed and started user operation in 2010. The system has been developed based on an automated data collection and data management architecture utilized for the confirmed scheme of SPring-8 mail-in data collection. Currently, further improvement to the remote access and automation which covers data processing and analysis are being developed.

X-ray crystal structure analysis of magnetically oriented microcrystals of lysozyme at 1.8 Å resolution

Microcrystals of lysozyme (5–10 µm) suspended in an ultraviolet-light-curable resin were aligned three dimensionally under a non-uniformly rotating magnetic field, and then the resin was consolidated by photopolymerization to obtain a composite in which microcrystals were three-dimensionally aligned. The obtained composite (MOMA: magnetically oriented microcrystal array) was analysed using a synchrotron X-ray source. A resolution of 1.8 A was obtained, which is in marked contrast to the resolution of 3 A previously reported for these composites, obtained by using an in-house X-ray diffractometer. These results show that a combination of MOMAs with conventional synchrotron beamlines may have valuable potential for crystal analyses of protein crystals that do not grow to larger sizes.

SPring-8 BL41XU, a high-flux macromolecular crystallography beamline.

SPring-8 BL41XU provides a high-flux X-ray beam of size 10–50 µm, and enables high-quality diffraction data to be obtained from various types of protein crystals. Details of this beamline and an upgrade project are described.

Hydration structures in proteins and neutron diffraction experiment on dissimilatory sulfite reductase D (DsrD)

Neutron crystallography can provide a substantial amount of information about the hydration of proteins. The hydration patterns of three proteins, whose structures have been solved at 1.5 or 1.6 A resolution using our BIX-type diffractometers, show interesting features. The water molecules adopt a variety of shapes in the neutron Fourier maps, revealing details of intermolecular hydrogen-bond formation and dynamics of hydration. In addition, the neutron diffraction study of a DNA-binding protein, dissimilatory sulfite reductase D (DsrD) is briefly described, and some preliminary results are presented. This topic is of interest because it is well known that hydrogen bonds play important roles in DNA-protein recognition.

Present status of SPring-8 macromolecular crystallography beamlines

Seven beamlines are operated for macromolecular crystallography (MX) at SPring‐8. The three undulator beamlines are developed for cutting edge target and four bending‐magnet beamlines are developed for high throughput MX. The undulator beamline, BL41XU that provides the most brilliant beam, is dedicated to obtain high quality data even from small‐size and weakly‐diffracting crystals. The minimum beam size at sample position is achieved to 10 μm diameter using a pin‐hole collimator. Its photon flux at wavelength λ = 1.0 A is 2.8×1011 photons/sec. This small beam coupled with irradiation point scanning method is quite useful to take diffraction dataset from small crystals by suppressing the radiation damage. These advanced technologies made a number of difficult protein structure analysis possible, (i.e. Sodium‐potassium ATPase). The bending‐magnet beamlines BL26B1/B2 and BL38B1 provide automatic data collection exploiting the high mobility of the beam. The beamline operation software “BSS,” sample auto‐cha...

Hydrogen bonds of DsrD protein revealed by neutron crystallography

Hydrogen bonds of DNA-binding protein DsrD have been determined by neutron diffraction. In terms of proton donors and acceptors, DsrD protein shows striking differences from other proteins.