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Gram-negative Sphingomonas sp. strain A1 exhibits positive chemotaxis toward acidic polysaccharide pectin. SPH1118 has been identified as a pectin-binding protein involved in both pectin chemotaxis and assimilation. Here we show tertiary structures of SPH1118 with six different conformations as determined by X-ray crystallography. SPH1118 consisted of two domains with a large cleft between the domains and substrates bound to positively charged and aromatic residues in the cleft through hydrogen bond and stacking interactions. Substrate-free SPH1118 adopted three different conformations in the open form. On the other hand, the two domains were closed in substrate-bound form and the domain closure ratio was changed in response to the substrate size, suggesting that the conformational change upon binding to the substrate triggered the expression of pectin chemotaxis and assimilation. This study first clarified that the solute-binding protein with dual functions recognized the substrate through flexible conformational changes in response to the substrate size.

Proper anther and pollen development are important for plant reproduction. The plant hormone gibberellin is important for anther development in rice, but its gametophytic functions remain largely unknown. Here, we report the functional and evolutionary analyses of rice gibberellin 3-oxidase 1 (OsGA3ox1), a gibberellin synthetic enzyme specifically expressed in the late developmental stages of anthers. Enzymatic and X-ray crystallography analyses reveal that OsGA3ox1 has a higher GA7 synthesis ratio than OsGA3ox2. In addition, we generate an osga3ox1 knockout mutant by genome editing and demonstrate the bioactive gibberellic acid synthesis by the OsGA3ox1 action during starch accumulation in pollen via invertase regulation. Furthermore, we analyze the evolution of Oryza GA3ox1s and reveal that their enzyme activity and gene expression have evolved in a way that is characteristic of the Oryza genus and contribute to their male reproduction ability., The authors solve the crystal structure of OsGA3ox2 and predict that of OsGA3ox1. These enzymes catalyze the final step in the biosynthesis of gibberellin, one of the plant hormones. Evolutionary analysis combined with the new structure reveal important aspects of the OsGA3ox1’s function in plant male reproduction.

X-ray structural analysis of gibberellin (GA) and auxin inactivation enzymes GA 2-oxidase 3 (GA2ox3) and auxin dioxygenase was used to elucidate the post-translational regulatory mechanism of homeostasis of growth phytohormones in rice. Results revealed that both enzymes form multimers by bridging their substrates, GA4 and indole acetic acid (IAA), at their interface regions. Further functional analyses revealed that multimerization of these enzymes gradually proceeds with increasing GA4 and IAA concentrations, and multimerized enzymes have much higher specific activities than monomer forms, a system that should favour homeostasis maintenance of these phytohormones. Molecular dynamic analysis explained the mechanism responsible for increasing GA2ox3 activity by multimerization—GA4 in the interface of oligomerized GA2ox3s can enter the active site with a low energy barrier. In summary, we report that the homeostatic systems for maintaining GA and IAA levels, based on the same allosteric mechanism, developed independently at the times when seed plants and angiosperms appeared.

Soybean mature glycinin was purified and crystallized and its preliminary crystallographic analysis is also reported., Glycinin is one of the most abundant storage-protein molecules in soybean seeds and is composed of five subunits (A1aB1b, A1bB2, A2B1a, A3B4 and A5A4B3). A1bB2 was purified from a mutant soybean cultivar containing glycinin composed of only A5A4B3 and A1bB2. At 281 K the protein formed hexagonal, rectangular and rod-shaped crystals in the first [0.1 M imidazole pH 8.0, 0.2 M MgCl2, 35%(v/v) MPD], second [0.1 M sodium citrate pH 5.6, 0.2 M ammonium acetate, 30%(v/v) MPD] and third (0.1 M phosphate–citrate pH 4.2, 2.0 M ammonium sulfate) crystallization conditions, respectively. X-ray diffraction data were collected to resolutions of 1.85, 1.85 and 2.5 Å from crystals of the three different shapes. The crystals belonged to space groups P6322, P21 and P1, with unit-cell parameters a = b = 143.60, c = 84.54 Å, a = 114.54, b = 105.82, c = 116.67 Å, β = 94.99° and a = 94.45, b = 94.96, c = 100.66 Å, α = 107.02, β = 108.44, γ = 110.71°, respectively. One, six and six subunits of A1bB2 were estimated to be present in the respective asymmetric units. The three-dimensional structure of the A1bB2 hexamer is currently being determined.

The covalent conjugation of a 14-carbon saturated fatty acid (myristic acid) to the amino-terminal glycine residue is critical for some viral proteins to function. This protein lipidation modification, termed N-myristoylation, is targeted by host cytotoxic T lymphocytes (CTLs) that specifically recognize N-myristoylated short peptides; however, the molecular mechanisms underlying lipopeptide antigen (Ag) presentation remain elusive. Here we show that a primate major histocompatibility complex (MHC) class I-encoded protein is capable of binding N-myristoylated 5-mer peptides and presenting them to specific CTLs. A high-resolution X-ray crystallographic analysis of the MHC class I:lipopeptide complex reveals an Ag-binding groove that is elaborately constructed to bind N-myristoylated short peptides rather than prototypic 9-mer peptides. The identification of lipopeptide-specific, MHC class I-restricted CTLs indicates that the widely accepted concept of MHC class I-mediated presentation of long peptides to CTLs may need some modifications to incorporate a novel MHC class I function of lipopeptide Ag presentation.

β-1,4-Mannanase (EC 3.2.1.78) catalyzes the hydrolysis of β-1,4-glycosidic bonds within mannan, a major constituent group of the hemicelluloses. Bivalves and gastropods possess β-1,4-mannanase and may degrade mannan in seaweed and/or phytoplankton to obtain carbon and energy using the secreted enzymes in their digestive systems. In the present study, the crystal structure of AkMan, a gastropod β-1,4-mannanase prepared from the common sea hare Aplysia kurodai, was determined at 1.05 Å resolution. This is the first report of the three-dimensional structure of a gastropod β-1,4-mannanase. The structure was compared with bivalve β-1,4-mannanase and the roles of residues in the catalytic cleft were investigated. No obvious binding residue was found in subsite +1 and the substrate-binding site was exposed to the molecular surface, which may account for the enzymatic properties of mannanases that can digest complex substrates such as glucomannan and branched mannan.

Sphingomonas sp. A1 directly incorporates alginate polysaccharides through a`superchannel' comprising a pit on the cell surface, alginate-binding proteins inthe periplasm and an ABC transporter (alginate importer) in the inner membrane. Alginate importer, consisting of four subunits, AlgM1, AlgM2 and two molecules of AlgS, was crystallized in the presence of the binding protein AlgQ2. Preliminary X-ray analysis showed that the crystal diffracted to 3.3 Å resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a=72.5, b = 136.8, c = 273.3 Å, suggesting the presence of one complex in the asymmetric unit.

Thaumatin, an intensely sweet-tasting plant protein, elicits a sweet taste at a concentration of 50 nM. The crystal structure of a recombinant form of thaumatin I produced in the yeast Pichia pastoris has been determined to a resolution of 1.1 Å. The model was refined with anisotropic B parameters and riding H atoms. A comparison of the diffraction data and refinement statistics for recombinant thaumatin I with those for plant thaumatin I revealed no significant differences in the diffraction data. The R values for recombinant thaumatin I and plant thaumatin I (F(o) > 4σ) were 9.11% and 9.91%, respectively, indicating the final model to be of good quality. Notably, the electron-density maps around Asn46 and Ser63, which differ between thaumatin variants, were significantly improved. Furthermore, a number of H atoms became visible in an OMIT map and could be assigned. The high-quality structure of recombinant thaumatin with H atoms should provide details about sweetness determinants in thaumatin and provide valuable insights into the mechanism of its interaction with taste receptors.

L-Azetidine-2-carboxylate hydrolase from Pseudomonas sp. strain A2C catalyzes a ring-opening reaction that detoxifies L-azetidine-2-carboxylate, an analogue of L-proline. Recombinant L-azetidine-2-carboxylate hydrolase was overexpressed, purified and crystallized using polyethylene glycol and magnesium acetate as precipitants. The needle-shaped crystal belonged to space group P2(1), with unit-cell parameters a = 35.6, b = 63.6, c = 54.7 A, beta = 105.5 degrees . The crystal diffracted to a resolution of 1.38 A. The calculated V(M) value was 2.2 A(3) Da(-1), suggesting that the crystal contains one enzyme subunit in the asymmetric unit.

Peanuts contain some of the most potent food allergens known to date. Ara h 1 is one of the three major peanut allergens. As a first step towards three-dimensional structure elucidation, recombinant Ara h 1 core region was cloned, expressed in Escherichia coli and purified to homogeneity. Crystals were obtained using 0.1 M sodium citrate pH 5.6, 0.1 M NaCl, 15% PEG 400 as precipitant. The crystals diffracted to 2.25 A resolution using synchrotron radiation and belonged to the monoclinic space group C2, with unit-cell parameters a=156.521, b=88.991, c=158.971 A, beta=107.144 degrees. Data were collected at the BL-38B1 station of SPring-8 (Hyogo, Japan).

本研究ではスペースシャトル｢エンデバー｣(STS-47)を利用してニワトリ卵白リゾチーム、ウマ骨格筋ミオグロビン、緑膿菌ω-アミノ酸:ピルビン酸アミノ基転移酵素、クモノスカビのリパーゼ、およびヒトのインシュリンの5種類のタンパク質または酵素について微小重力下、20度Cで6種のタンパク質結晶化に関する宇宙実験を実施した。結晶化方法は静置バッチ法を採用し、この原理に基づいて、宇宙実験装置として密閉系のポリカーボネート製酵素結晶化容器を製作した。6種のタンパク質試料のうち、インシュリン以外の5種の試料について結晶が得られ、そのうち4種の試料が単結晶であった。リゾチーム(pH4.5)とω-アミノ酸:ピルビン酸アミノ基転移酵素は結晶学的に良質の大きな単結晶に成長し、筑波の高エネルギー物理学研究所の放射光実験施設(PF)のシンクロトロン放射光を利用してX線結晶学的解析を実施した。, The space experiment on the crystallization of six proteins was carried out at 20 C in a microgravity state of space shuttle 'Endeavor' (STS-47). The proteins or enzymes used include fowl albumen lysozyme, horse skeletal muscle myoglobin, Pseudomonas aeruginosa omega-amino acid: pyruvic acid aminotransferase, spiderweb fungal lipase, and human insulin. The stationary batch method was used for crystallization. Based on its principle, a closed system polycarbonate vessel for enzyme crystallization was made as a space experiment device. Five of the six protein samples, except for human insulin, provided crystallized samples. Four of the five samples were single crystals. Lysozyme (pH 4.5) and omega-amino acid: pyruvic acid aminotransferase grew to crystallographically good and large single crystals. They were X-ray crystallographically analyzed using synchrotron radiation of the High Energy Physics Laboratory's Photon Factory (PF) in Tsukuba., 資料番号: AA0004115006, レポート番号: NASDA-TMR-940002 V.1