Takae Yamauchi

Crystal Structure of a Homolog of Mammalian Serine Racemase from Schizosaccharomyces pombe

d-Serine is an endogenous coagonist for the N-methyl-d-aspartate receptor and is involved in excitatory neurotransmission in the brain. Mammalian pyridoxal 5′-phosphate-dependent serine racemase, which is localized in the mammalian brain, catalyzes the racemization of l-serine to yield d-serine and vice versa. The enzyme also catalyzes the dehydration of d- and l-serine. Both reactions are enhanced by Mg·ATP in vivo. We have determined the structures of the following three forms of the mammalian enzyme homolog from Schizosaccharomyces pombe: the wild-type enzyme, the wild-type enzyme in the complex with an ATP analog, and the modified enzyme in the complex with serine at 1.7, 1.9, and 2.2 Å resolution, respectively. On binding of the substrate, the small domain rotates toward the large domain to close the active site. The ATP binding site was identified at the domain and the subunit interface. Computer graphics models of the wild-type enzyme complexed with l-serine and d-serine provided an insight into the catalytic mechanisms of both reactions. Lys-57 and Ser-82 located on the protein and solvent sides, respectively, with respect to the cofactor plane, are acid-base catalysts that shuttle protons to the substrate. The modified enzyme, which has a unique “lysino-d-alanyl” residue at the active site, also exhibits catalytic activities. The crystal-soaking experiment showed that the substrate serine was actually trapped in the active site of the modified enzyme, suggesting that the lysino-d-alanyl residue acts as a catalytic base in the same manner as inherent Lys-57 of the wild-type enzyme.

A redox-active columnar metallomesogen and its cyclic voltammetric responses

We demonstrate a strategy that appears particularly well suited for the design of redox-active liquid crystals and also constitutes the first demonstration of cyclic voltammetric responses of a metallomesogen in the columnar liquid crystalline phase.

A facile and versatile preparation of bilindiones and biladienones from tetraarylporphyrins

Bilindiones and biladienones carrying aryl groups at the meso positions were prepared using coupled oxidation reactions of iron tetraarylporphyrins in 20-63% yield.

The crystal structure of maleylacetate reductase from Rhizobium sp. strain MTP‐10005 provides insights into the reaction mechanism of enzymes in its original family

Maleylacetate reductase plays a crucial role in catabolism of resorcinol by catalyzing the NAD(P)H‐dependent reduction of maleylacetate, at a carbon–carbon double bond, to 3‐oxoadipate. The crystal structure of maleylacetate reductase from Rhizobium sp. strain MTP‐10005, GraC, has been elucidated by the X‐ray diffraction method at 1.5 Å resolution. GraC is a homodimer, and each subunit consists of two domains: an N‐terminal NADH‐binding domain adopting an α/β structure and a C‐terminal functional domain adopting an α‐helical structure. Such structural features show similarity to those of the two existing families of enzymes in dehydroquinate synthase‐like superfamily. However, GraC is distinct in dimer formation and activity expression mechanism from the families of enzymes. Two subunits in GraC have different structures from each other in the present crystal. One subunit has several ligands mimicking NADH and the substrate in the cleft and adopts a closed domain arrangement. In contrast, the other subunit does not contain any ligand causing structural changes and adopts an open domain arrangement. The structure of GraC reveals those of maleylacetate reductase both in the coenzyme, substrate‐binding state and in the ligand‐free state. The comparison of both subunit structures reveals a conformational change of the Tyr326 loop for interaction with His243 on ligand binding. Structures of related enzymes suggest that His243 is likely a catalytic residue of GraC. Mutational analyses of His243 and Tyr326 support the catalytic roles proposed from structural information. The crystal structure of GraC characterizes the maleylacetate reductase family as a third family in the dehydroquinate synthase‐like superfamily. Proteins 2016; 84:1029–1042.

Crystallographic studies of aspartate racemase from Lactobacillus sakei NBRC 15893

Aspartate racemase catalyzes the interconversion between L-aspartate and D-aspartate and belongs to the PLP-independent racemases. The enzyme from the lactic acid bacterium Lactobacillus sakei NBRC 15893, isolated from kimoto, is considered to be involved in D-aspartate synthesis during the brewing process of Japanese sake at low temperatures. The enzyme was crystallized at 293 K by the sitting-drop vapour-diffusion method using 25%(v/v) PEG MME 550, 5%(v/v) 2-propanol. The crystal belonged to space group P3121, with unit-cell parameters a = b = 104.68, c = 97.29 Å, and diffracted to 2.6 Å resolution. Structure determination is under way.

Crystal structure analysis of L-asparaginase from Thermococcus litoralis DSM 5473

L-Asparaginase (E.C. 3.5.1.1) is an enzyme that catalyzes the hydrolysis of L-asparagine to L-aspartate and ammonia. The enzyme has been used as an effective antineoplastic agent for acute lymphoblastic leukemia. In food industry, it is expected as a food processing aid to reduce acrylamide levels in cooked food. Thermococcus litoralis DSM 5473, which was isolated from a hot spring of a shallow sea, is a hyperthermophilic archaeon and has the ability to grow optimally at 88 °C. In order to study the industrial utilization of thermostable L-asparaginase, the gene encoding the enzyme from T. litoralis DSM 5473 (TlASNase) was cloned and expressed in Escherichia coli. TlASNase has a distinctive substrate specificity; the enzymatic activity to D-Asn is 70% of that to L-Asn whereas other reported L-asparaginases have no or low activity to D-Asn. To elucidate the structural basis for the catalytic mechanism, substrate recognition, and thermostability, we analyzed the crystal structure of TlASNase. The enzyme was crystallized at 20 °C by the sitting-drop vapour-diffusion method using 25% (v/v) 2-propanol, 0.25 M ammonium acetate, and 0.1 M Tris-HCl pH 8.5. Diffraction measurement was performed at beamline BL17A, Photon Factory, Tsukuba, Japan. During the measurement the crystal soaked in crystallization solution containing 30% (v/v) glycerol was cooled in a nitrogen stream 100 K. The crystal belonged to space group P6122 with unit cell parameters a = b = 69.55, c = 558.15 Å and diffracted to 2.3 Å resolution. The structure was determined by molecular replacement and refined at 2.3 Å resolution. TlASNase is a homodimer, existing in the asymmetric unit of the crystal. The subunit has 331 amino acid residues and consists of two domains connected by a linker region of 20 amino acid residues. The N-terminal domain of 185 amino acid residues adopts a large α/β-structure, which comprises four α-helices and twelve β-strands. Its core structure is flavodoxin-like fold with four α-helices and eight β-strands. The C-terminal domain of 126 amino acid residues has a small α/β-structure, which has a parallel β-sheet of four β-strands with five α-helices. The TlASNase dimer is formed mainly by intersubunit interactions between β-strands of C-terminal domains, resulting in the formation of the large intersubunit eight stranded β-sheet. From the amino-acid sequence alignment and structural comparison with related enzymes, Thr12 and Thr86 in TlASNase are supposed to be catalytic residues. The structural comparison of active sites shows that the two portions of the loop regions in the active site have different conformations from those of related enzymes. These sites are candidates for the recognition sites to substrates. The trials of the crystallization of the enzymesubstrate/product complexes are under way.

Crystal structure analyses of oxygenase component of resorcinol hydroxylase

The resorcinol hydroxylase is involved in the first step of the resorcinol catabolic pathway and catalyzes hydroxylation of resorcinol to hydroxyquinol. The enzyme belongs to the two-component flavin-diffusible monooxygenase family and acts in the coexistence of two components: an oxygenase and a flavin reductase. The oxygenase component hydroxylates the substrate using molecular oxygen and reduced flavin produced by the reductase. To understand the structural basis for the catalytic mechanism, we analyzed the crystal structure of the oxygenase component (GraA) from Rhizobium sp. strain MTP-10005. The GraA subunit has 409 amino acid residues. Apo-form crystals were obtained in the tetragonal space group I4122 by a sitting-drop vapor-diffusion method with a reservoir solution of PEG3350 and K2HPO4. Holo-form crystals were obtained in the trigonal space group P3221 by a sitting-drop vapordiffusion method with a reservoir solution of PEG3350 and KNO3. Both structures were determined by molecular replacement and refined at 2.3 Å and 3.2 Å resolutions, respectively. GraA is a homotetramer with three molecular two-fold axes identical to crystallographic two-fold axes in the apo-form crystal. In the holo-form crystal, four tetramers exist in the asymmetric unit and each subunit binds one FAD. The subunit consists of three domains. The N-terminal domain has an α-structure mainly of antiparallel αhelices; the central domain has a β-structure of two β-sheets stacked together; the C-terminal domain has a four-helix-bundle structure of long antiparallel α-helices involved in tetramer formation. In the holo-form, the FAD is located in the space that is encompassed by these three domains. The loop region of 13 residues, which is disordered in the apo-form, is ordered and covers FAD of another subunit. The turn portion of the loop occludes the entrance of the putative active site.

Crystal structures of bacterial flavin reductase GraD and its complex with NADH

Rhizobium sp. strain MTP-10005 uses the aromatic compound γ-resorcylate as a sole source of carbon and energy for growth. Resorcinol hydroxylase, which converts resorcinol to hydroxyquinol, plays an important role in the aerobic microbial catabolism of γresorcylate. Resorcinol hydroxylase from Rhizobium sp. strain MTP-10005 is a two-component enzyme consisting of the reductase and the monooxygenase components. The reductase component (GraD) is an oxidoreductase containing a flavin molecule as a cofactor. GraD catalyzes the NADH-dependent reduction of free FAD according to a ping-pong bisubstrate-biproduct mechanism. The reduced FAD is then used by the monooxygenase component GraA to hydroxylate resorcinol to hydroxyquinol. We have determined the three-dimensional structures of recombinant GraD with a bound FAD and in complex with NAD. GraD was crystallized at 293 K by the sitting-drop vapour-diffusion method using a precipitant solution containing 13 14% (w/v) PEG 2000, 6 9% (v/v) 2-propanol, 100 mM sodium citrate pH 5.6, 100 mM DTT and 200 μM FAD. The approximate dimensions of the obtained crystals were 0.1 × 0.1 × 0.15 mm3. The crystal diffracted to 1.8Å and belongs to space group P41212 with unit-cell parameters of a = b = 77.8 Å and c = 124.2 Å. The crystal structure has been determined by the molecular replacement and refined at 1.8 Å resolution. GraD exists as a homodimer, and each monomer contains an FAD. The probable binding site for NADH is covered with the N-terminal sub-domain in chain A, whereas the site is completely exposed to bulk solvent in chain B. The NAD-complex crystals were prepared by soaking the GraD crystals in the reservoir solution supplemented with NADH. The crystal diffracted to 1.8 Å, and the crystal structure was determined at 1.8 Å resolution. The Fo-Fc maps for the crystal soaked with NADH showed the electron densities corresponding to the nicotinamide ring and the adenyl moiety in chain B.

Structural features and low-temperature adaptation of aspartate racemase

Aspartate racemase (AspR) catalyzes the interconversion between Land D-aspartates without PLP. The only crystal structure of the PLP-independent amino-acid racemase is now available from a hyperthermophilic archaeon. To elucidate structural features and lowtemperature adaptation of the racemase group, we determined the crystal structures of AspR from Lactobacillus sakei NBRC 15893 (LsAspR), which works in the low-to-medium temperature range, and for comparison AspR from Thermococcus litoralis DSM 5473 (TlAspR), which has the maximum activity at 95 °C. LsAspR and TlAspR weree crystallized at 20 °C by the sitting-drop vapour-diffusion method using a precipitant solution of 25% (v/v) PEG-MME 550, 5% (v/v) 2-propanol and 0.1 M sodium acetate pH 4.8 and a precipitant solution of 24% (w/v) PEG1500, 0.2 M L-proline and 0.1 M HEPES pH 7.5, respectively. The structures of LsAspR and TlAspR were determined by molecular replacement and refined at 2.6 Å resolution (R=23.8%, Rfree = 31.6%) and 2.0 Å resolution (R=18.7%, Rfree = 25.0%), respectively. Both LsAspR and TlAspR molecules are homodimers with molecular two-fold axis. The subunit of each enzyme molecule comprises the N-terminal and C-terminal domains. The molecule is formed mainly by intersubunit interactions between the N-terminal α-helices and intersubunit hydrogen-bonds between the N-terminal β-sheets in the dimer interface. The active-site cleft exists between both the domains. The spatial arrangement of the strictly conserved cystein residues in the cleft reveals the Cys residues involved in the enzymatic catalysis. A structural comparison of LsAspR and TlAspR reveals structural factors probably involved in thermostability of AspR. The molecular volume, intersubunit interaction, and the number of ion pairs suggest that the LsAspR molecule is more loose than that of TlAspR.