Hajime Sugawara

Atomic Structure of Plant Glutamine Synthetase: A KEY ENZYME FOR PLANT PRODUCTIVITY

Plants provide nourishment for animals and other heterotrophs as the sole primary producer in the food chain. Glutamine synthetase (GS), one of the essential enzymes for plant autotrophy catalyzes the incorporation of ammonia into glutamate to generate glutamine with concomitant hydrolysis of ATP, and plays a crucial role in the assimilation and re-assimilation of ammonia derived from a wide variety of metabolic processes during plant growth and development. Elucidation of the atomic structure of higher plant GS is important to understand its detailed reaction mechanism and to obtain further insight into plant productivity and agronomical utility. Here we report the first crystal structures of maize (Zea mays L.) GS. The structure reveals a unique decameric structure that differs significantly from the bacterial GS structure. Higher plants have several isoenzymes of GS differing in heat stability and catalytic properties for efficient responses to variation in the environment and nutrition. A key residue responsible for the heat stability was found to be Ile-161 in GS1a. The three structures in complex with substrate analogues, including phosphinothricin, a widely used herbicide, lead us to propose a mechanism for the transfer of phosphate from ATP to glutamate and to interpret the inhibitory action of phosphinothricin as a guide for the development of new potential herbicides.

Crystal structure of the histidine-containing phosphotransfer protein ZmHP2 from maize.

In higher plants, histidine‐aspartate phosphorelays (two‐component system) are involved in hormone signaling and stress responses. In these systems, histidine‐containing phosphotransfer (HPt) proteins mediate the signal transmission from sensory histidine kinases to response regulators, including integration of several signaling pathways or branching into different pathways. We have determined the crystal structure of a maize HPt protein, ZmHP2, at 2.2 Å resolution. ZmHP2 has six α‐helices with a four‐helix bundle at the C‐terminus, a feature commonly found in HPt domains. In ZmHP2, almost all of the conserved residues among plant HPt proteins surround this histidine, probably forming the docking interface for the receiver domain of histidine kinase or the response regulator. Arg102 of ZmHP2 is conserved as a basic residue in plant HPt proteins. In bacteria, it is replaced by glutamine or glutamate that form a hydrogen bond to Nδ atoms of the phospho‐accepting histidine. It may play a key role in the complex formation of ZmHP2 with receiver domains.

Amino Acid Sequence and Carbohydrate-binding Analysis of the N-acetyl- D -galactosamine-specific C-Type Lectin, CEL-I, from the Holothuroidea, Cucumaria echinata

CEL-I is one of the Ca2+-dependent lectins that has been isolated from the sea cucumber, Cucumaria echinata. This protein is composed of two identical subunits held by a single disulfide bond. The complete amino acid sequence of CEL-I was determined by sequencing the peptides produced by proteolytic fragmentation of S-pyridylethylated CEL-I. A subunit of CEL-I is composed of 140 amino acid residues. Two intrachain (Cys3-Cys14 and Cys31-Cys135) and one interchain (Cys36) disulfide bonds were also identified from an analysis of the cystine-containing peptides obtained from the intact protein. The similarity between the sequence of CEL-I and that of other C-type lectins was low, while the C-terminal region, including the putative Ca2+ and carbohydrate-binding sites, was relatively well conserved. When the carbohydrate-binding activity was examined by a solid-phase microplate assay, CEL-I showed much higher affinity for N-acetyl-D-galactosamine than for other galactose-related carbohydrates. The association constant of CEL-I for p-nitrophenyl N-acetyl-β-D-galactosaminide (NP-GalNAc) was determined to be 2.3×104 M1, and the maximum number of bound NP-GalNAc was estimated to be 1.6 by an equilibrium dialysis experiment.

Structural insight into the reaction mechanism and evolution of cytokinin biosynthesis

The phytohormone cytokinin regulates plant growth and development. This hormone is also synthesized by some phytopathogenic bacteria, such as Agrobacterium tumefaciens, and is as a key factor in the formation of plant tumors. The rate-limiting step of cytokinin biosynthesis is catalyzed by adenosine phosphate-isopentenyltransferase (IPT). Agrobacterium IPT has a unique substrate specificity that enables it to increase trans-zeatin production by recruiting a metabolic intermediate of the host plants biosynthetic pathway. Here, we show the crystal structures of Tzs, an IPT from A. tumefaciens, complexed with AMP and a prenyl-donor analogue, dimethylallyl S-thiodiphosphate. The structures reveal that the carbon-nitrogen-based prenylation proceeds by the SN2-reaction mechanism. Site-directed mutagenesis was used to determine the amino acid residues, Asp-173 and His-214, which are responsible for differences in prenyl-donor substrate specificity between plant and bacterial IPTs. IPT and the p loop-containing nucleoside triphosphate hydrolases likely evolved from a common ancestral protein. Despite structural similarities, IPT has evolved a distinct role in which the p loop transfers a prenyl moiety in cytokinin biosynthesis.

X-ray structure of Galdieria Rubisco complexed with one sulfate ion per active site.

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the reactions of carboxylation and oxygenation of ribulose‐1,5‐bisphosphate. These reactions require that the active site should be closed by a flexible loop (loop 6) of the large subunit. Rubisco from a red alga, Galdieria partita, has the highest specificity for carboxylation reaction among the Rubiscos hitherto reported. The crystal structure of unactivated Galdieria Rubisco has been determined at 2.6 Å resolution. The electron density map reveals that a sulfate binds only to the P1 anion‐binding site of the active site and the loop 6 is closed. Galdieria Rubisco has a unique hydrogen bond between the main chain oxygen of Val332 on the loop 6 and the ϵ‐amino group of Gln386 of the same large subunit. This interaction is likely to be crucial to understanding for stabilizing the loop 6 in the closed state and to making a higher affinity for anionic ligands.

Crystallization and preliminary X-ray analysis of a bacterial lysozyme produced by Streptomyces globisporus

The extracellular bacteriolytic enzyme produced by Streptomyces globisporus shows a beta-1,4-N,6-O-diacetylmuramidase activity as well as a beta-1,4-N-acetylmuramidase activity. Crystals of this enzyme have been obtained by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. They belong to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = 63.11 (4), c = 121.1 (1) A, diffract to at least 2.0 A resolution and are suitable for high-resolution structure analysis. The crystal structure was solved by molecular replacement using lysozyme produced by S. erythraeus as a search model. The structure refinement is now in progress.

Crystallization and preliminary X-ray diffraction study of the histidine-containing phosphotransfer protein ZmHP1 from maize.

In histidine-aspartate phosphorelays (two-component systems) involved in plant-hormone signalling, histidine-containing phosphotransfer (HPt) proteins mediate the transfer of a phosphoryl group from the sensory histidine kinase to the response regulator. The maize HPt protein ZmHP1 has been crystallized. Although ZmHP1 with an N-terminal His tag could be crystallized using sodium chloride as a precipitant, the crystals diffracted poorly to only 3.2 A resolution. When the His tag was removed, ZmHP1 crystals were obtained using polyethylene glycol 4000 as a precipitant and the diffraction data were greatly enhanced to 2.4 A resolution. The crystals belonged to the space group P4(1)2(1)2, with one ZmHP1 molecule in the asymmetric unit.

Crystallization and preliminary crystallographic study of an invertebrate C-type lectin, CEL-I, from the marine invertebrate Cucumaria echinata

CEL-I is a GalNAc-specific carbohydrate-binding protein (lectin) isolated from the sea cucumber Cucumaria echinata. This protein belongs to the widely distributed C-type lectin family of animal lectins, which require Ca(2+) for their carbohydrate-binding ability and play important roles in various molecular-recognition processes in organisms. CEL-I was crystallized with 2-methyl-2,4-pentanediol using the hanging-drop vapour-diffusion technique. The CEL-I crystals belong to the monoclinic space group C2, with unit-cell parameters a = 92.38 (3), b = 69.94 (3), c = 76.69 (3) A, beta = 136.46 (2) degrees. Diffraction data were collected to 2.0 A resolution using synchrotron radiation. The asymmetric unit contains one CEL-I molecule.

Crystallization and preliminary X-ray analysis of plastocyanin from cyanobacterium Synechococcus sp. PCC 7942

A plastocyanin from the cyanobacterium Synechococcus sp. PCC 7942 has been crystallized in two different forms by hanging-drop vapour diffusion with ammonium sulfate as precipitant. Form I is hexagonal, space group P61 or P65, with unit-cell dimensions a = b = 34.62 and c = 107.22 A. Form II is tetragonal, space group P41 or P43, with unit-cell dimensions a = b = 43.05 and c = 56.94 A. Form I crystals diffract to 2.5 A using graphite-monochromated Cu Kalpha radiation from a Rigaku RU-300 rotating-anode generator operated at 40 kV and 100 mA. Form II crystals diffract to 1.9 A using synchrotron radiation at beamline BL6A of the Photon Factory (KEK). Molecular-replacement calculations using the structure of plastocyanin from Ulva pertusa have been performed.

Crystal Structure of Rubisco from a Thermophilic Red Alga, Galdieria Partita

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO, EC 4.1.1.39) is the initial step enzyme in the Calvin-Benson cycle of photosynthesis. It catalyzes the addition of gaseous CO2 to ribulose 1,5-bisphosphate (RuBP) and produces two molecules of 3-phosphoglycerate (3-PGA) (1,2). However, this enzyme also catalyzes O2 addition for RuBP as the primary reaction of photorespiration. The reaction yields one molecule each of 3-PGA and 2-phosphoglycolate from RuBP. The oxygenation reaction impairs the photosynthetic efficiency by up to 60% (3). Thus the improvement of carboxylation/oxygenation ratio by genetic engineering has been attempted to increase the productivity of crop plants.