Atomic Structure of Plant Glutamine Synthetase

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. 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. Inorganic nitrogen is an essential, often limiting nutrient for plant growth and development. In most natural soils, nitrate is the major form of inorganic nitrogen. After uptake of nitrate, plants first reduce it to ammonia, and then assimilate it into an organic compound as an amide moiety of glutamine. Because glutamine synthetase (GS) 7The abbreviations used are: GS, glutamine synthetase; MetSox-P, methionine sulfoximine phosphate; PEG, polyethylene glycol; MetSox, methionine sulfoximine; PPT-P, phosphinothricin phosphate; AMPPNP, adenylyl imidodiphosphate; PPT, phosphinothricin; NCS, non-crystallographic symmetry. 7The abbreviations used are: GS, glutamine synthetase; MetSox-P, methionine sulfoximine phosphate; PEG, polyethylene glycol; MetSox, methionine sulfoximine; PPT-P, phosphinothricin phosphate; AMPPNP, adenylyl imidodiphosphate; PPT, phosphinothricin; NCS, non-crystallographic symmetry. catalyzes the very step of assimilation of inorganic nitrogen and because the amide moiety of glutamine is utilized as the donor of amino residue to synthesize a number of essential metabolites such as amino acids, nucleic acids, and amino sugars, glutamine synthesis by plant GS is the cornerstone of plant productivity and thus nitrogen nourishment of all animals on the Earth. For this reason, the importance of plant GS is comparable with that of ribulose-1,5-bisphosphate carboxylase/oxygenase, the carbon dioxide assimilating enzyme (1Buchanan B.B. Gruissem W. Jones R.L. Biochemistry & Molecular Biology of Plants. John Wiley & Sons, New York2000Google Scholar). Comparison of the primary structures of GSs from prokaryotes and eukaryotes, results in plant GS being categorized as type II, this type commonly occurring in eukaryotes including animals (2Kumada Y. Benson D.R. Hillemann D. Hosted T.J. Rochefort D.A. Thompson C.J. Wohlleben W. Tateno Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3009-3013Crossref PubMed Scopus (179) Google Scholar). In contrast, type I GS is widely found in prokaryotes (2Kumada Y. Benson D.R. Hillemann D. Hosted T.J. Rochefort D.A. Thompson C.J. Wohlleben W. Tateno Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3009-3013Crossref PubMed Scopus (179) Google Scholar). The regulatory mechanisms of type I GS activity such as adenylylation and metabolite feedback have been thoroughly characterized (3Rhee S.G. Chock P.B. Stadtman E.R. Adv. Enzymol. Relat. Areas Mol. Biol. 1989; 62: 37-92PubMed Google Scholar). The crystal structures of GS from Mycobacterium tuberculosis (4Krajewski W.W. Jones T.A. Mowbray S.L. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10499-10504Crossref PubMed Scopus (68) Google Scholar) and Salmonella typhimurium (5Almassy R.J. Janson C.A. Hamlin R. Xuong N.H. Eisenberg D. Nature. 1986; 323: 304-309Crossref PubMed Scopus (231) Google Scholar) have been determined and the proteins shown to be dodecameric, with each dodecamer being composed of one identical subunit with a molecular mass of about 52 kDa. Types I and II GSs are thought to share a common ancestor but to have diverged into the two types at a very early stage during molecular evolution (2Kumada Y. Benson D.R. Hillemann D. Hosted T.J. Rochefort D.A. Thompson C.J. Wohlleben W. Tateno Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3009-3013Crossref PubMed Scopus (179) Google Scholar). Only a faint similarity in the primary structures is appreciable between the two types of GS and it seems that no sophisticated regulatory mechanism as seen in Type I GS exists in Type II GS. As no three-dimensional structure of any plant or animal GS has yet been determined, our structural understanding of Type II GS still remains obscure.Plant GSs are divided into two subtypes with different subcellular localization: GS1 in the cytosol and GS2 in the plastids. GS1 is encoded by a small multigene family, and the GS1 members are further categorized into two groups based on expression profile in response to external nitrogen status, enzymatic property, and physicochemical stability. Maize has five GS1s (GS1a-GS1e) (6Sakakibara H. Kawabata S. Takahashi H. Hase T. Sugiyama T. Plant Cell Physiol. 1992; 33: 49-58Google Scholar, 7Sakakibara H. Shimizu H. Hase T. Yamazaki Y. Takao T. Shimonishi Y. Sugiyama T. J. Biol. Chem. 1996; 271: 29561-29568Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), and two representative isoforms that are well characterized namely GS1a and GS1d, show high sequence identity (86%), but show remarkable difference in stability (7Sakakibara H. Shimizu H. Hase T. Yamazaki Y. Takao T. Shimonishi Y. Sugiyama T. J. Biol. Chem. 1996; 271: 29561-29568Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). We chose GS1a for an initial trial for three-dimensional structure determination because of its high stability (7Sakakibara H. Shimizu H. Hase T. Yamazaki Y. Takao T. Shimonishi Y. Sugiyama T. J. Biol. Chem. 1996; 271: 29561-29568Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). We report the role of Ile-161 responsible for differences in heat stability between the two GS isozymes, and three complex structures of maize GS1a revealing the reaction mechanism of phosphate transfer and inhibitory mechanism of PPT used as a herbicide.EXPERIMENTAL PROCEDURESPreparation of Recombinant Maize GS Proteins—Escherichia coli strain JM109 cells transformed with GS1a, GS1d, and various mutant GS genes were grown in Luria broth supplemented with 50 μg/ml ampicillin for 2 h (4 h for crystallization) at 37 °C after inoculation with an overnight seed culture at 1% volume. Isopropyl β-d-thiogalactoside was then added to a final concentration of 1 mm, and cultivation was continued for a further 12 h. The cells were harvested by centrifugation at 4,000 × g for 10 min and stored at -30 °C until use. Maize GS1a purification for crystallization were performed as described previously (7Sakakibara H. Shimizu H. Hase T. Yamazaki Y. Takao T. Shimonishi Y. Sugiyama T. J. Biol. Chem. 1996; 271: 29561-29568Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar).Crystallization—GS crystals were grown at 20 °C in 2-3 weeks by the hanging-drop vapor diffusion method. GS crystals in complex with ADP and methionine sulfoximine phosphate (MetSox-P) were obtained by mixing 4 μl of protein solution (17.5 mg/ml3) with 4 μl of reservoir solution (9% (w/v) polyethylene glycol 8000 (PEG8000, Hampton Research), 100 mm Tris-HCl (pH 7.8), 5% (v/v) 2-methyl-2,4-pentanediol, 10 mm MnCl2) containing 1 mm ATP and 1 mm MetSox. GS crystals in complex with ADP and phosphinothricin phosphate (PPT-P) and with AMPPNP and MetSox were obtained the same way as above except using a reservoir solution containing 1 mm ATP and 1 mm PPT, and 1 mm AMPPNP and 1 mm MetSox, respectively. The crystals were harvested in a solution comprising 17.5% (w/v) PEG8000, 100 mm Tris-HCl (pH 7.8), 5% (v/v) 2-methyl-2,4-pentanediol, 10 mm MnCl2, and 1 mm substrate analogues. Heavy atom derivatives were obtained by soaking native crystals for 12 h in the harvest solution containing 1 mm K2PtCl4 or 1mm HgCl2. Each crystal was mounted in a glass capillary before data collection.Structure Determination—Diffraction data, except native data set II, were collected in-house with a Rigaku FR-E Super Bright rotating anode x-ray generator at a wavelength of 1.5418 Å (CuKα) using a RAXIS VII (Rigaku) imaging plate detector. High resolution data (native II) were collected using synchrotron radiation on beamline BL5A at the Photon Factory (Tsukuba, Japan) with a CCD detector Quantum 315 (ADSC, Poway, CA). All data collections were performed at a temperature of 295 K because cryocooling of crystals degraded diffraction resolution. Indexing, integration, and scaling of all diffraction data sets were performed using the program HKL2000 (8Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38368) Google Scholar). Data collection statistics are summarized in Table 1. Native I, mercury, and platinum data sets were used for phase calculation by multiple isomorphous replacement with anomalous scattering using the program SOLVE (9Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D. 1999; 55: 849-861Crossref PubMed Scopus (3219) Google Scholar). Density modification, model building, and refinement were done with RESOLVE (10Terwilliger T.C. Acta Crystallogr. Sect. D. 2000; 56: 965-972Crossref PubMed Scopus (1631) Google Scholar, 11Terwilliger T.C. Acta Crystallogr. Sect. D. 2003; 59: 38-44Crossref PubMed Scopus (593) Google Scholar), O (12Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13004) Google Scholar), and REFMAC5 (13Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (13779) Google Scholar). In subsequent refinement, native data set I was replaced with native data set II. NCS restraints were applied among 10 subunits throughout refinement. ADP, MetSox-P, and Mn2+ ion models were fitted into the substrate binding sites based on the difference electron density map (Fig. 1). Three Mn2+ positions per subunit were confirmed in NCS averaged anomalous difference Fourier maps (Fig. 1) from CuKα radiation. Omit maps for residues 202-237 in chain A were depicted in Supplementary Fig. S1. Figs. 2, 4, and 7A were prepared with MOLSCRIPT (14Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar) and RASTER3D (15Merritt E.A. Murphy M.E. Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 869-873Crossref PubMed Scopus (2854) Google Scholar). Fig. 3, a and b, were prepared with PyMOL. Structure-based sequence alignment with M. tuberculosis GS was performed using the program MATRAS (16Kawabata T. Nucleic Acids Res. 2003; 31: 3367-3369Crossref PubMed Scopus (216) Google Scholar).TABLE 1Data collection and refinement statisticsCrystal typeADP/MetSox-P/MnADP/PPT-P/MnAMPPNP/MetSox/MnData collection and processing statisticsData setNative IINative IHgPtSpace groupP21P21P21P21P21P21Unit cell dimension (Å)a (Å)95.895.195.195.896.295.8b (Å)191.0190.6190.7191.0191.0190.9c (Å)118.1118.1118.1118.2118.2117.9β (°)101.5101.5101.3101.4101.3101.2Wavelength (Å)1.00001.54181.54181.54181.54181.5418Resolution (Å)50.0-2.6322.0-3.230.0-3.750.0-3.350-3.850-3.5Measured334,295165,797182,850336,77558,501146,178I/σI15.8 (2.1)8.4 (3.1)13.3 (5.3)16.7 (4.5)5.8 (1.8)12.0 (3.2)Redundancy3.12.54.25.42.83.4Completeness (%)87.1 (65)98.1 (99.1)99.0 (100)99.6 (100)82.5 (77.1)83.1 (87.2)RmergeaRmerge = 100 ∑|I - 〈I 〉|/∑ I, where I is the observed intensity and 〈I 〉 is the average intensity of multiple observations of symmetry-related reflections. (%)6.4 (38)9.0 (26)12.2 (26)9.3 (32)14.5 (35.5)9.5 (29.3)Phasing statisticsRisobRiso = 100 ∑||FPH| - |FP||/∑|FP|, where |FP| and |FPH| are the observed native and heavy-atom derivative structure factor amplitudes, respectively. (%)19.5 (21.4)16.5 (22.5)No. of sites1010Refinement statisticsResolution (Å)26.13-2.6327.36-3.8133.50-3.50Protein atoms27,46027,46027,460Ligand atoms450450450Water molecule721238377Rwork/Rfree (%)18.4/21.918.4/22.916.5/20.9Root mean square deviationsBond lengths (Å)0.0160.0200.015Bond angles (°)1.6691.7631.616a Rmerge = 100 ∑|I - 〈I 〉|/∑ I, where I is the observed intensity and 〈I 〉 is the average intensity of multiple observations of symmetry-related reflections.b Riso = 100 ∑||FPH| - |FP||/∑|FP|, where |FP| and |FPH| are the observed native and heavy-atom derivative structure factor amplitudes, respectively. Open table in a new tab FIGURE 2Structure of maize GS1a. a, a top view of the overall structure from the direction of the NCS 5-fold symmetry. Each subunit is shown in a different color. b, a side view of the overall structure from the direction of the NCS 2-fold symmetry. c, arrangement of two neighboring subunits within one ring. The two subunits are colored red and orange and their N-terminal and C-terminal domains are shown in light and deep coloring, respectively. The active site is formed between the C-terminal domain of one subunit and the N-terminal domain of the other subunit. d, interaction of two subunits. The side chain of Ile-161 of one subunit is in close proximity (within 4 Å) to the side chains of Leu-33, Tyr-219, Arg-223, and Glu-222 of the adjacent subunit.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Representation of the interactions between enzyme and substrate analogues. a, stick models for the interaction of the enzyme with AMPPNP and MetSox. Carbon, oxygen, nitrogen, phosphorus, and sulfur atoms are colored gray, red, blue, salmon, and yellow, respectively. Three Mn2+ are indicated in pink spheres. Dotted lines designate hydrogen bonds and coordination bonds to Mn2+ ions. Residues without dotted lines have hydrophobic interactions with the substrate. b, stick models for the interaction of the enzyme with ADP and MetSox. c, stick models for the interaction of the enzyme with ADP and PPT-P.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 7Active site structure formed by Asp-56 and Glu-297 of maize GS1a. a, stereo view of the side chain structures of Asp-56′ and Glu-297 together with ADP, MetSox-P, and three Mn2+ ions. b, alignments of short stretches of amino acid sequences containing Asp-56 and Glu-297 in GSs from M. tuberculosis (MtGS), S. typhimurium (StGS), maize (GS1 isozymes GS1a, GS1d, andGS2), Arabidopsis, human, and chicken.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Comparison of oligomeric structures of plant and bacterial GSs. a, molecular anatomy of the decameric structure of maize GS1a. The original structure is graphically separated into two pentameric rings to show the side view of the two rings. Each ring is further rotated by 90 degrees to show the inner and outer surfaces of the ring. In comparison of the primary structures of maize GS1a and M. tuberculosis GS (see panel c of this figure), the regions present in both the enzymes are colored blue and insertion regions unique to maize GS1a are colored white. Residues located at the contact area of two rings are colored yellow. b, molecular anatomy of the dodecameric structure of M. tuberculosis GS. The same graphical processing and coloring as used for the plant GS are employed to compare the oligomerization status of the two enzymes. c, structural alignment of amino acid sequences of maize GS1a and M. tuberculosis GS. Residues identical between the two enzymes are boxed and the residues located in the contact areas of the two rings are shown in red.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Comparison of oligomeric structures of plant and bacterial GSs. a, molecular anatomy of the decameric structure of maize GS1a. The original structure is graphically separated into two pentameric rings to show the side view of the two rings. Each ring is further rotated by 90 degrees to show the inner and outer surfaces of the ring. In comparison of the primary structures of maize GS1a and M. tuberculosis GS (see panel c of this figure), the regions present in both the enzymes are colored blue and insertion regions unique to maize GS1a are colored white. Residues located at the contact area of two rings are colored yellow. b, molecular anatomy of the dodecameric structure of M. tuberculosis GS. The same graphical processing and coloring as used for the plant GS are employed to compare the oligomerization status of the two enzymes. c, structural alignment of amino acid sequences of maize GS1a and M. tuberculosis GS. Residues identical between the two enzymes are boxed and the residues located in the contact areas of the two rings are shown in red.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Construction of GS1a-GS1d Chimera and Site-directed Mutagenes-is—E. coli JM105 (supE endA sbcB15 hsdR4 rpsL thi Δ(lac-proAB) F′[traD36 proAB+ lacIq lacZ ΔM15]) was used for the construction of plasmids carrying GS1a-GS1d chimeric genes. Plasmid pTrcGS1a-GS1d was constructed from pTrcGS1a and pTrcGS1d (7Sakakibara H. Shimizu H. Hase T. Yamazaki Y. Takao T. Shimonishi Y. Sugiyama T. J. Biol. Chem. 1996; 271: 29561-29568Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In pTrcGS1a-GS1d, GS1a and GS1d genes are located in tandem in the same orientation under control of the Ptrc promoter. The plasmid was linearized by digestion with KpnI and EcoRI, which are located between the two GS genes, and transformed to E. coli JM105. The transformants were incubated on a Luria-agar plate containing 50 μg/ml ampicillin. Only recombined and circularized plasmid(s) were rescued by the ampicillin selection. The colonies were randomly selected and restriction analysis of the chimeric GS gene was performed to classify the recombined sites. Finally, nucleotide sequencing of the clones was performed to determine the recombined site.GS mutants that have one amino acid substitution were constructed by an overlap extension method by two-step polymerase chain reaction using a combination of two terminal primers and a pair of two mutagenic primers. All mutation sites and the sequence integrity of the entire coding region of GS were confirmed by DNA sequencing.Enzyme Assay—The preparation of crude extracts of E. coli cells was performed as described previously (7Sakakibara H. Shimizu H. Hase T. Yamazaki Y. Takao T. Shimonishi Y. Sugiyama T. J. Biol. Chem. 1996; 271: 29561-29568Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The activities of GS were determined by the methods of Cullimore and Sims (17Cullimore J.V. Sims A.P. Planta. 1980; 150: 392-396Crossref PubMed Scopus (86) Google Scholar). One unit of GS activity was defined as the amount of enzyme that produced 1 μmol of γ-glutamylhydroxamate per min under the conditions of the assays of GS activities.RESULTS AND DISCUSSIONOverall Structure—Three different crystal forms of maize GS1a in complex with ADP and glutamate analogues were prepared in the presence of Mn2+, a divalent cation essential for GS activity (3Rhee S.G. Chock P.B. Stadtman E.R. Adv. Enzymol. Relat. Areas Mol. Biol. 1989; 62: 37-92PubMed Google Scholar). Substrate combinations were ADP and Met-Sox-P (18Ronzio R.A. Rowe W.B. Meister A. Biochemistry. 1969; 8: 1066-1075Crossref PubMed Scopus (253) Google Scholar) (ADP/MetSox-P/Mn), AMPPNP and MetSox (AMPPNP/MetSox/Mn), and ADP and PPT-P (19Logusch E.W. Walker D.M. McDonald J.F. Franz J.E. Biochemistry. 1989; 28: 3043-3051Crossref PubMed Scopus (43) Google Scholar) (ADP/PPT-P/Mn). Crystal structures of the three GS1a derivatives were determined at 2.63-, 3.50-, and 3.80-Å resolutions, respectively (Table 1). Electron density maps in these complex data sets were extremely clear (supplementary Fig. S1, Fig. 1) considering resolution limits, revealing features of most side chains and the detail of the substrate/substrate analogue structures for the crystals of AMPPNP/MetSox/Mn and ADP/PPT-P/Mn. High quality electron density maps in all complex data sets led to the final Rfree and Rwork values almost equal to that of high resolution crystal analysis (Table 1), hence allowing our detailed discussion of the reaction mechanism for phosphate transfer described below.The maize GS crystal structure is decamer with dimensions of 115 Å × 115 Å × 95 Å, having 52 symmetry with five 2-fold axes perpendicular to a 5-fold axis (Fig. 2, a and b). The decameric structure is composed of two face-to-face pentameric rings of identical subunits, with a total of 10 active sites, each formed between every two neighboring subunits within each ring (Fig. 2a).The early study indicated the octamer structure for eukaryote GS derived from electron microscope observations, which reported that one octamer structure was composed of two tetramer rings (20Tsuprun V.L. Samsonidze T.G. Radukina N.A. Pushkin A.V. Evstigneeva Z.G. Kretovich W.L. Biochim. Biophys. Acta. 1980; 626: 1-4Crossref PubMed Scopus (9) Google Scholar, 21Haschemeyer R.H. Trans. N. Y. Acad. Sci. 1968; 30: 875-891Crossref PubMed Scopus (17) Google Scholar). We previously reported a solution experiment (7Sakakibara H. Shimizu H. Hase T. Yamazaki Y. Takao T. Shimonishi Y. Sugiyama T. J. Biol. Chem. 1996; 271: 29561-29568Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), in which the gel filtration elution pattern of maize GS1a showed a single peak estimating a total molecular mass at 440 kDa in solution. The total molecular mass of decameric maize GS1a without metal ions or substrates/substrate analogues is 398 kDa, which approximately correspond to that estimated with gel filtration. The structural description only from our crystal structure analysis may raise a possibility of pentamer structure in solution, and of an artificial decameric formation from crystal packing. The solution experiment supports the decamer structure in solution in which the accessible surface area between the two pentamer is small (933 Å) but is thought to be sufficiently plausible.Subunit Structure—The structure of one subunit consists of a smaller N-terminal domain (residues 1-103) and a larger C-terminal domain (residues 104-356), with the N-terminal domain of one subunit and the C-terminal domain of the neighboring subunit forming an active site (Fig. 2c). Structure-based sequenced alignment with M. tuberculosis GS shows sequence identity of 22.2% (Fig. 3c) and secondary structure identity of 84.2% (α-helix, β-strand, or coil) for structurally corresponding 356 residues, whereas the root mean square deviation of the 356 Cα atoms between aligned residues is moderate (3.92 Å), indicating the two structures have diverged from the common ancestor. The overall fold of the maize GS structure is similar to those of M. tuberculosis and S. typhimurium, but there are several large differences described as follows. Residues of M. tuberculosis GS lacking their counterparts in maize GS are mainly located at the molecular surface of M. tuberculosis GS. The C-terminal residues (residues 393-478) of M. tuberculosis lacking their counterparts in maize GS play a role as the adenylylation site and contribute to intimate interactions between two hexamer rings (helical thong). Residues 143-154 of M. tuberculosis GS lacking in maize GS are those of the β-loop contributing to the hydrophobic interactions of the two hexamer rings. The conformations of maize GS and M. tuberculosis GS differ significantly for residues of 1-18 (1-17) and 145-152 (158-180) with residue ranges in parentheses for M. tuberculosis GS. Most of residues for binding substrate and divalent ions are conserved between maize and bacterial GS and hence the enzyme reaction mechanism of maize GS is likely to be essentially the same as that of bacterial GS. Each active site contains three Mn2+ atoms in the middle of the catalytic cleft (Fig. 2c).Structural Difference between Plant and Bacterial GSs—Remarkable differences between plant and bacterial GSs are notable in the mode of inter-ring subunit contact. Interface surface area between the two pentameric rings of plant GS is 933 Å2, which is about 17 times smaller than that of the hexameric rings of bacterial GS (17,238 Å2) (Fig. 3, a and b). The inter-ring interaction is achieved by five pairs of two subunits, whose centroids are apart by 67.5 degrees with 5-fold axis (Fig. 2, a and b), and thus subunit interaction becomes very limited: only 4 hydrophobic and 2 hydrogen bonding interactions (supplementary Fig. S2a). In contrast, the inter-ring interaction of the bacterial enzyme is much stronger: centroids of interacting subunits are totally overlapped with 6-fold axis and the two subunits are stabilized by 37 hydrophobic and 36 hydrogen bonding interactions contributed by 43 residues (supplementary Fig. S2b). Plant GS has several internal deletions and a C-terminal truncation in comparison with the bacterial enzyme (Fig. 3c). Such structural differences occurring during molecular evolution have resulted in the appearance of unique quaternary structures, which have given rise to fundamental differences in the ways of regulation of activity of type I and type II GSs.Substrate Binding Site—The GS decamer contains 10 active sites and each active site is located between two adjacent subunits in a pentamer. The active sites (20 Å deep) are formed between two neighboring monomers in a ring where its opening is roughly in parallel to the 5-fold axis (Fig. 2a). Well defined electron densities even without NCS averaging for each of ADP, AMPPNP, Mn2+, MetSox, phosphorylated MetSox, and phosphorylated PPT was found in all substrate binding sites in a decamer, respectively (Fig. 1). The ADP binding sites in the ADP/MetSox-P/Mn and ADP/PPT-P/Mn complex structures, and the AMPPNP binding sites in the AMPPNP/MetSox/Mn complex structure each are located near the openings in the 10 catalytic clefts. The phosphorylated MetSox (P-MetSox) binding sites were found at the bottoms of the 10 clefts. Three Mn2+ ions lie at the middle of the cleft. Three Mn2+ ions are called Mn-n1, Mn-n2, and Mn-n3, respectively, corresponding to the numbering of M. tuberculosis by Krajewski et al. (4Krajewski W.W. Jones T.A. Mowbray S.L. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10499-10504Crossref PubMed Scopus (68) Google Scholar) (Fig. 4). The positions of the three Mn2+ were confirmed by CuKα anomalous difference Fourier maps (Fig. 1). The ADP molecule in the ADP/MetSox-P/Mn complex are bound by Gly-127, Ser-187, Asn-251, Ser-253, Tyr-328, and Arg-316, through hydrophobic and hydrogen bonding interactions, and its β-phosphate is bound by Mn-n2 and Mn-n3 through coordination bond interactions (Mn-n2 and Mn-n3 coordinate phosphate oxygen atoms) (Fig. 4b). The P-MetSox molecule is bound mainly by the main chain of Gly-245 and the side chains of Glu-131, Glu-192, His-249, Arg-291, Arg-311, and Arg-332 through hydrogen bond interactions in addition to three Mn2+ ions (Fig. 4b). The phosphate group of the P-MetSox coordinates to the three Mn2+. The P-PPT is bound by the same protein residues and three Mn2+ ions as those of the P-MetSox (Fig. 4c). Interactions between AMPPNP and protein residues in the active site are ap