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The 3D structure of Taka-amylase A was determined by X-ray crystal analysis at 3 Å resolution by Masao Kakudo’s laboratory at the Institute for Protein Research, Osaka University, in 1980. Seven kinds of heavy atom derivatives were used for phase determination. There are three copies of Taka-amylase molecules in the asymmetric unit, which improved the quality of electron density maps, leading to the completion of a molecular model with 478 amino acids. The structure determination process in those days is described briefly.

Starch produced by plants is a stored form of energy and is an important dietary source of calories for humans and domestic animals. Disproportionating enzyme (D-enzyme) catalyzes intramolecular and intermolecular transglycosylation reactions of α-1, 4-glucan. D-enzyme is essential in starch metabolism in the potato. We present the crystal structures of potato D-enzyme, including two different types of complex structures: a primary Michaelis complex (substrate binding mode) for 26-meric cycloamylose (CA26) and a covalent intermediate for acarbose. Our study revealed that the acarbose and CA26 reactions catalyzed by potato D-enzyme involve the formation of a covalent intermediate with the donor substrate. HPAEC of reaction substrates and products revealed the activity of the potato D-enzyme on acarbose and CA26 as donor substrates. The structural and chromatography analyses provide insight into the mechanism of the coupling reaction of CA and glucose catalyzed by the potato D-enzyme. The enzymatic reaction mechanism does not involve residual hydrolysis. This could be particularly useful in preventing unnecessary starch degradation leading to reduced crop productivity. Optimization of this mechanism would be important for improvements of starch storage and productivity in crops.

To investigate the mechanism for low pH adaptation by a carboxylesterase, structural and biochemical analyses of EstFa_R (a recombinant, slightly acidophilic carboxylesterase from Ferroplasma acidiphilum) and SshEstI (an alkaliphilic carboxylesterase from Sulfolobus shibatae DSM5389) were performed. Although a previous proteomics study by another group showed that the enzyme purified from F. acidiphilum contained an iron atom, EstFa_R did not bind to iron as analyzed by inductively coupled plasma MS and isothermal titration calorimetry. The crystal structures of EstFa_R and SshEstI were determined at 1.6- and 1.5-Å resolutions, respectively. EstFa_R had a catalytic triad with an extended hydrogen bond network that was not observed in SshEstI. Quadruple mutants of both proteins were created to remove or introduce the extended hydrogen bond network. The mutation on EstFa_R enhanced its catalytic efficiency and gave it an alkaline pH optimum, whereas the mutation on SshEstI resulted in opposite effects (i.e. a decrease in the catalytic efficiency and a downward shift in the optimum pH). Our experimental results suggest that the low pH optimum of EstFa_R activity was a result of the unique extended hydrogen bond network in the catalytic triad and the highly negatively charged surface around the active site. The change in the pH optimum of EstFa_R happened simultaneously with a change in the catalytic efficiency, suggesting that the local flexibility of the active site in EstFa_R could be modified by quadruple mutation. These observations may provide a novel strategy to elucidate the low pH adaptation of serine hydrolases.

In eukaryotic DNA replication initiation, hexameric MCM (mini-chromosome maintenance) unwinds the template double-stranded DNA to form the replication fork. MCM is activated by two proteins, Cdc45 and GINS, which constitute the 'CMG' unwindosome complex together with the MCM core. The archaeal DNA replication system is quite similar to that of eukaryotes, but only limited knowledge about the DNA unwinding mechanism is available, from a structural point of view. Here, we describe the crystal structure of an archaeal GAN (GINS-associated nuclease) from Thermococcus kodakaraensis, the homolog of eukaryotic Cdc45, in both the free form and the complex with the C-terminal domain of the cognate Gins51 subunit (Gins51C). This first archaeal GAN structure exhibits a unique, 'hybrid' structure between the bacterial RecJ and the eukaryotic Cdc45. GAN possesses the conserved DHH and DHH1 domains responsible for the exonuclease activity, and an inserted CID (CMG interacting domain)-like domain structurally comparable to that in Cdc45, suggesting its dual roles as an exonuclease in DNA repair and a CMG component in DNA replication. A structural comparison of the GAN-Gins51C complex with the GINS tetramer suggests that GINS uses the mobile Gins51C as a hook to bind GAN for CMG formation.

The structure of acetamidase/formamidase (Amds/Fmds) from the archaeon Thermoanaerobacter tengcongensis has been determined by X-ray diffraction analysis using MAD data in a crystal of space group P2₁, with unit-cell parameters a = 41.23 (3), b = 152.88 (6), c = 100.26 (7) Å, β = 99.49 (3) ° and been refined to a crystallographic R-factor of 17.4% and R-free of 23.7%. It contains two dimers in one asymmetric unit, in which native Amds/Fmds (TE19) contains of the 32 kDa native protein. The final model consists of 4 monomer (299 amino acids residues with additional 2 expression tag amino acids residues), 5 Ca²⁺, 4 Zn²⁺ and 853 water molecules. The monomer is composed by the following: an N-domain which is featuring by three-layers β/β/β; a prominent excursion between N-terminal end of strand β₇ and β₁₁, which contains four-stranded antiparallel β sheet; an C-domain which is formed by the last 82 amino acid residues with the feature of mixed α/β structure. The protein contains ion-pair Ca²⁺-Zn²⁺. The portion of three-layer β/β/β along with the loops provides four protein ligands to the tightly bound Ca²⁺, three water molecules complete the coordination; and provides five protein ligands to the tightly bound Zn²⁺, one water molecule complete the coordination.

Members of the genus Actinoplanes are considered to be representative of motile actinomycetes. To infer the flagellar diversity of Actinoplanes species, novel degenerate primers were designed for the flagellin (fliC) gene. The fliC gene of 21 Actinoplanes strains was successfully amplified and classified into two groups based on whether they were large (type I) or small (type II). Comparison of the translated amino acid sequences revealed that this size difference could be attributed to large number of gaps located in the central variable region. However, the C- and N- terminal regions were conserved. Except for a region on the flagellum surface, structural predictions of type I and II flagellins revealed that the two flagellin types were strongly correlated with each other. Phylogenetic analysis of the 115-amino acid N-terminal sequences revealed that the Actinoplanes species formed three clusters, and type II flagellin gene containing three type strains were phylogenetically closely related each other.

The development of a stable L-BDH chimera was attempted by exchanging whole domains between two native structural analogs, L-BDH and meso-BDH, because the S-configuration specificity of L-BDH is valuable from the standpoint of its application but its activity is unstable, whereas meso-BDH is stable. The domain chimeras obtained indicated that the leaf-like structures constituting three domains were likely to be mainly associated with chiral recognition, and the fourth domain, the basic domain, is likely to be mainly associated with enzyme stability. A combination of the leaf domains of L-BDH and the basic domain of meso-BDH attained a sufficient level of practical use as an artificial L-BDH chimera, because the resulting enzyme had both stability and S-configuration specificity. However, the levels of stability and specificity were slightly lower than those of the respective enzymes from which they were derived.

The structures of isomaltase from Saccharomyces cerevisiae and in complex with maltose were determined at resolutions of 1.30 and 1.60 A, respectively. Isomaltase contains three domains, namely, A, B, and C. Domain A consists of the (β/α)8-barrel common to glycoside hydrolase family 13. However, the folding of domain C is rarely seen in other glycoside hydrolase family 13 enzymes. An electron density corresponding to a nonreducing end glucose residue was observed in the active site of isomaltase in complex with maltose; however, only incomplete density was observed for the reducing end. The active site pocket contains two water chains. One water chain is a water path from the bottom of the pocket to the surface of the protein, and may act as a water drain during substrate binding. The other water chain, which consists of six water molecules, is located near the catalytic residues Glu277 and Asp352. These water molecules may act as a reservoir that provides water for subsequent hydrolytic events. The best substrate for oligo-1,6-glucosidase is isomaltotriose; other, longer-chain, oligosaccharides are also good substrates. However, isomaltase shows the highest activity towards isomaltose and very little activity towards longer oligosaccharides. This is because the entrance to the active site pocket of isomaltose is severely narrowed by Tyr158, His280, and loop 310–315, and because the isomaltase pocket is shallower than that of other oligo-1,6-glucosidases. These features of the isomaltase active site pocket prevent isomalto-oligosaccharides from binding to the active site effectively.

Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes.

The BAHD family is a class of acyl-CoA-dependent acyltransferases that are involved in plant secondary metabolism and show a diverse range of specificities for acyl acceptors. Anthocyanin acyltransferases make up an important class of the BAHD family and catalyze the acylation of anthocyanins that are responsible for most of the red-to-blue colors of flowers. Here, we describe crystallographic and mutational studies of three similar anthocyanin malonyltransferases from red chrysanthemum petals: anthocyanidin 3-O-glucoside-6''-O-malonyltransferase (Dm3MaT1), anthocyanidin 3-O-glucoside-3'', 6''-O-dimalonyltransferase (Dm3MaT2), and a homolog (Dm3MaT3). Mutational analyses revealed that seven amino acid residues in the N- and C-terminal regions are important for the differential acyl-acceptor specificity between Dm3MaT1 and Dm3MaT2. Crystallographic studies of Dm3MaT3 provided the first structure of a BAHD member, complexed with acyl-CoA, showing the detailed interactions between the enzyme and acyl-CoA molecules. The structure, combined with the results of mutational analyses, allowed us to identify the acyl-acceptor binding site of anthocyanin malonyltransferases, which is structurally different from the corresponding portion of vinorine synthase, another BAHD member, thus permitting the diversity of the acyl-acceptor specificity of BAHD family to be understood.

When expressed in Escherichia coli , cytosolic poplar glutaredoxin C1 (CGYC active site) exists as a dimeric iron–sulfur-containing holoprotein or as a monomeric apoprotein in solution. Analytical and spectroscopic studies of wild-type protein and site-directed variants and structural characterization of the holoprotein by using x-ray crystallography indicate that the holoprotein contains a subunit-bridging [2Fe–2S] cluster that is ligated by the catalytic cysteines of two glutaredoxins and the cysteines of two glutathiones. Mutagenesis data on a variety of poplar glutaredoxins suggest that the incorporation of an iron–sulfur cluster could be a general feature of plant glutaredoxins possessing a glycine adjacent to the catalytic cysteine. In light of these results, the possible involvement of plant glutaredoxins in oxidative stress sensing or iron–sulfur biosynthesis is discussed with respect to their intracellular localization.

The chaperone system is known to be exploited by viruses for their replication. In the present study, we identified the cochaperone FKBP6 as a host factor required for hepatitis C virus (HCV) replication. FKBP6 is a peptidyl prolyl cis-trans isomerase with three domains of the tetratricopeptide repeat (TPR), but lacks FK-506 binding ability. FKBP6 interacted with HCV nonstructural protein 5A (NS5A) and also formed a complex with FKBP6 itself or FKBP8, which is known to be critical for HCV replication. The Val121 of NS5A and TPR domains of FKBP6 were responsible for the interaction between NS5A and FKBP6. FKBP6 was colocalized with NS5A, FKBP8 and double-stranded RNA in HCV-infected cells. HCV replication was completely suppressed in FKBP6-knockout hepatoma cell lines, while the expression of FKBP6 restored HCV replication in FKBP6-knockout cells. A treatment with the FKBP8 inhibitor N-(N′, N′-dimethylcarboxamidomethyl)cycloheximide impaired the formation of a homo- or hetero-complex consisting of FKBP6 and/or FKBP8 and suppressed HCV replication. HCV infection promoted the expression of FKBP6, but not that of FKBP8, in cultured cells and human liver tissue. These results indicate that FKBP6 is an HCV-induced host factor that supports viral replication in cooperation with NS5A.

The human malaria parasite (Plasmodium falciparum) possesses a plastid-derived organelle called the apicoplast, which is believed to employ metabolisms crucial for the parasite's survival. We cloned and studied the biochemical properties of plant-type ferredoxin (Fd) and Fd-NADP+ reductase (FNR), a redox system that potentially supplies reducing power to Fd-dependent metabolic pathways in malaria parasite apicoplasts. The recombinant P. falciparum Fd and FNR proteins were produced by synthetic genes with altered codon usages preferred in Escherichia coli. The redox potential of the Fd was shown to be considerably more positive than those of leaf-type and root-type Fds from plants, which is favourable for a presumed direction of electron flow from catabolically generated NADPH to Fd in the apicoplast. The backbone structure of P. falciparum Fd, as solved by X-ray crystallography, closely resembles those of Fds from plants, and the surface-charge distribution shows several acidic regions in common with plant Fds and some basic regions unique to this Fd. P. falciparum FNR was able to transfer electrons selectively to P. falciparum Fd in a reconstituted system of NADPH-dependent cytochrome c reduction. These results indicate that an NADPH-FNR-Fd cascade is operative in the apicoplast of human malaria parasites.

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.

International audience; Glutaredoxins are glutathione-dependent enzymes that function to reduce disulfide bonds in vivo. Interestingly, a recent discovery indicates that some glutaredoxins can also exist in another form, an iron-sulfur protein [Lillig, C. H., et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 8168-8173]. This provides a direct connection between glutaredoxins and iron-sulfur proteins, suggesting a possible new regulatory role of iron-sulfur clusters along with the new functional switch of glutaredoxins. Biochemical studies have indicated that poplar glutaredoxin C1 (Grx-C1) is also such a biform protein. The apo form (monomer) of Grx-C1 is a regular glutaredoxin, and the holo form (dimer) is an iron-sulfur protein with a bridging [2Fe-2S] cluster. Here, we report the structural characterizations of poplar Grx-C1 in both the apo and holo forms by NMR spectroscopy. The solution structure of the reduced apo Grx-C1, which is the first plant Grx structure, shows a typical Grx fold. When poplar Grx-C1 forms a dimer with an iron-sulfur cluster, each subunit of the holo form still retains the overall fold of the apo form. The bridging iron-sulfur cluster in holo Grx-C1 is coordinated near the active site. In addition to the iron-sulfur cluster linker, helix 3 of each subunit is probably involved in the direct contact between the two subunits. Moreover, two glutathione molecules are identified in the vicinity of the iron-sulfur cluster and very likely participate in cluster coordination. Taken together, we propose that the bridging [2Fe-2S] cluster is coordinated by the first cysteine at the glutaredoxin active site from each subunit of holo Grx-C1, along with two cysteines from two glutathione molecules. Our studies reveal that holo Grx-C1 has a novel structural and iron-sulfur cluster coordination pattern for an iron-sulfur protein.

The three dimensional structures of the C(1)X(2)G (3)(X(3))X(4)C(5) motif of hCG, which is considered to be important for noncovalent assembly of the alpha- and beta-subunits of glycoprotein hormones were analyzed to assess the importance of glycine (Gly) (G) at site X(3) in the motif by the conformational energy calculation using computational procedures. In the C(1)M(2)G (3)(X(3))C(4)C(5) motif of the alpha-subunit, Ramachandran plot analysis showing the allowed area of the dihedral angles demonstrated that only a Gly residue was allowed at site X(3). In calculating collision with surrounding atoms as a monomer the possible main chain models of the C(1)A(2)G(3)(X(3))Y(4)C(5) motif in the beta-subunit showed that only alanine (Ala) (A) or Gly at site X(3) is allowed to alleviate the collision with the cysteine (Cys) (C) residues which form a disulfide bridge. A mutant of the beta-subunit with the C(1)A(2)A(3)(X(3))Y(4)C(5) motif (Ala at site X(3)) may not compose a heterodimer with the alpha-subunit because of interference of intermolecular hydrogen bond formation. These findings indicate that the Gly residue at site X(3) (G(3)) in the motif is essential for heterodimer formation of glycoprotein hormones. The significance of similar motifs found in various human proteins other than glycoprotein hormones was suggested.

CEL-III is a Ca2+-dependent animal lectin from the sea cucumber, Cucumaria echinata, which shows binding specificity for galactose-containing carbohydrates and exhibits hemolytic and hemagglutinating activities. We have determined its crystal structure at 1.7 A resolution by using single isomorphous replacement with anomalous scattering of lead. CEL-III consists of N-terminal two carbohydrate-binding domains and a C-terminal domain, which is essential for oligomerization and binding to a membrane. CEL-III is the first structure with two β-trefoil folds containing five Ca2+ ions. Here, we report the structural features of CEL-III and discuss the mechanisms of recognition of carbohydrates and hemolysis.

We have determined the crystal structure, at 1.2-A resolution, of Equisetum arvense ferredoxin isoform II (FdII), which lacks residues equivalent to Arg(39) and Glu(28) highly conserved among other ferredoxins (Fds). In other Fds these residues form an intramolecular salt bridge crucial for stabilization of the [2Fe-2S] cluster, which is disrupted upon complex formation with Fd-NADP(+) oxidoreductase (FNR) to form two intermolecular salt bridges. The overall structure of FdII resembles the known backbone structures of E. arvense isoform I (FdI) and other plant-type Fds. Dramatically, in the FdII structure a unique, alternative salt bridge is formed between Arg(22) and Glu(58). This results in a different relative orientation of the alpha-helix formed by Leu(23)-Glu(29) and eliminates the possibility of forming three of the five intermolecular salt bridges identified on formation of a complex between maize FdI and maize FNR. Mutation of FdII, informed by structural differences with FdI, showed that the alternative salt bridge and the absence of an otherwise conserved Tyr residue are important for the alternative stabilization of the FdII [2Fe-2S] cluster. We also investigated FdI and FdII electron transfer to FNR on chloroplast thylakoid membranes. The K(m) and V(max) values of FdII are similar to those of FdI, contrary to previous measurements of the reverse reaction, from FNR to Fd. The affinity between reduced FdI and oxidized FNR is much greater than that between oxidized FdI and reduced FNR, whereas this is not the case with FdII. The pH dependence of electron transfer by FdI, FdII, and an FdII mutant with FdI features was measured and further indicated that the binding mode to FNR differs between FdI and FdII. Based on this evidence, we hypothesize that binding modes with other Fd-dependent reductases may also vary between FdI and FdII. The structural differences between FdI and FdII therefore result in functional differences that may influence partitioning of electrons into different redox metabolic pathways.

CEL-I is a C-type lectin, purified from the sea cucumber Cucumaria echinata, that shows a high specificity for N-acetylgalactosamine (GalNAc). We determined the crystal structures of CEL-I and its complex with GalNAc at 2.0 and 1.7 A resolution, respectively. CEL-I forms a disulfide-linked homodimer and contains two intramolecular disulfide bonds, although it lacks one intramolecular disulfide bond that is widely conserved among various C-type carbohydrate recognition domains (CRDs). Although the sequence similarity of CEL-I with other C-type CRDs is low, the overall folding of CEL-I was quite similar to those of other C-type CRDs. The structure of the complex with GalNAc revealed that the basic recognition mode of GalNAc was very similar to that for the GalNAc-binding mutant of the mannose-binding protein. However, the acetamido group of GalNAc appeared to be recognized more strongly by the combination of hydrogen bonds to Arg115 and van der Waals interaction with Gln70. Mutational analyses, in which Gln70 and/or Arg115 were replaced by alanine, confirmed that these residues contributed to GalNAc recognition in a cooperative manner.

This research was originally published in Journal of Biological Chemistry. Tatsuya Uchida, Takayuki Yamasaki, Seiichiro Eto, Hajime Sugawara, Genji Kurisu, Atsushi Nakagawa, Masami Kusunoki and Tomomitsu Hatakeyama. Crystal structure of the hemolytic lectin CEL-III isolated from the marine invertebrate Cucumaria echinata : Implications of domain structure for its membrane pore-formation mechanism. Journal of Biological Chemistry. 2004; 279, 37133-37141. © the American Society for Biochemistry and Molecular Biology., CEL-III is a Ca^＋ -dependent and galactose-specific lectin purified from the sea cucumber, Cucumaria echinata, which exhibits hemolytic and hemagglutinating activities. Six molecules of CEL-III are assumed to oligomerize to form an ion-permeable pore in the cell membrane. We have determined the crystal structure of CEL-III by using single isomorphous replacement aided by anomalous scattering in lead at 1.7 Å resolution. CEL-III consists of three distinct domains as follows: the N-terminal two carbohydrate-binding domains (1 and 2), which adopt β-trefoil folds such as the B-chain of ricin and are members of the (QXW)_3 motif family; and domain 3, which is a novel fold composed of two α-helices and one β-sandwich. CEL-III is the first Ca^ -dependent lectin structure with two β-trefoil folds. Despite sharing the structure of the B-chain of ricin, CEL-III binds five Ca^ ions at five of the six subdomains in both domains 1 and 2. Considering the relatively high similarity among the five subdomains, they are putative binding sites for galactose-related carbohydrates, although it remains to be elucidated whether bound Ca^ is directly involved in interaction with carbohydrates. The paucity of hydrophobic interactions in the interfaces between the domains and biochemical data suggest that these domains rearrange upon carbohydrate binding in the erythrocyte membrane. This conformational change may be responsible for oligomerization of CEL-III molecules and hemolysis in the erythrocyte membranes.

CEL-III is a Ca(2+)-dependent and galactose-specific lectin purified from the sea cucumber, Cucumaria echinata, which exhibits hemolytic and hemagglutinating activities. Six molecules of CEL-III are assumed to oligomerize to form an ion-permeable pore in the cell membrane. We have determined the crystal structure of CELIII by using single isomorphous replacement aided by anomalous scattering in lead at 1.7 A resolution. CEL-III consists of three distinct domains as follows: the N-terminal two carbohydrate-binding domains (1 and 2), which adopt beta-trefoil folds such as the B-chain of ricin and are members of the (QXW)(3) motif family; and domain 3, which is a novel fold composed of two alpha-helices and one beta-sandwich. CEL-III is the first Ca(2+)-dependent lectin structure with two beta-trefoil folds. Despite sharing the structure of the B-chain of ricin, CEL-III binds five Ca(2+) ions at five of the six subdomains in both domains 1 and 2. Considering the relatively high similarity among the five subdomains, they are putative binding sites for galactose-related carbohydrates, although it remains to be elucidated whether bound Ca(2+) is directly involved in interaction with carbohydrates. The paucity of hydrophobic interactions in the interfaces between the domains and biochemical data suggest that these domains rearrange upon carbohydrate binding in the erythrocyte membrane. This conformational change may be responsible for oligomerization of CEL-III molecules and hemolysis in the erythrocyte membranes.

During the evolution of higher-plant root and leaf-type-specific Fd : FNR complexes from an original cyanobacterial type progenitor, rearrangement of molecular interaction has altered the relative orientation of prosthetic groups and there have been changes in complex induced conformational change. Selection has presumably worked on mutation of residues responsible for interaction between the two proteins, favoring optimized electron flow in a specific direction, and efficient dissociation following specific oxidation of leaf Fd and reduction of root Fd. Major changes appear to be: loss in both leaf and root complexes of a cyanobacterial mechanism that ensures Fd dissociation from the complex following change in Fd redox state, development of a structural rearrangement of Fd on binding to leaf FNR that results in a negative shift in Fd redox potential favorable to photosynthetic electron flow, creation of a vacant space in the root Fd:FNR complex that may allow access to the redox centers of other enzymes to ensure efficient channeling of heterotrophic reductant into bioassimilation. Further structural analysis is essential to establish how root type FNR distinguishes between Fd isoforms, and discover how residues not directly involved in intermolecular interactions may affect complex formation.

The crystal and molecular structure of a new diterpenyl glycoside, Ptr-1, is presented. The crystal is triclinic, space group P1, with a = 8.2414(8) A, b = 13.0826(9) A, c = 6.1427(8) A, α = 95.345(9)°, β = 111.589(8)°, γ = 96.726(7)°, V = 604.9(1) A3, Z = 1. The structure was solved by direct methods and refined by full-matrix least-squares methods to a final R = 0.049 (Rw = 0.099) for 1872 independent reflections. The molecular structure is based on a glycopyranosyl group and a tetracyclic group. The glycopyranosyl group is β-D-allopyranosyl group. The tetracyclic group consists of 1 five-membered and 3 six-membered rings.

A chimeric (2S, 3S)-butanediol dehydrogenase (cLBDH) was engineered to have the strict (S)-configuration specificity of the (2S, 3S)-BDH (BsLBDH) derived from Brevibacterium saccharolyticum as well as the enzymatic stability of the (2R, 3S)-BDH (KpMBDH) from Klebsiella pneumonia by swapping the domains of two native BDHs. However, while cLBDH possesses the stability, it lacks the specificity. In order to assist in the design a BDH having strict substrate specificity, an X-ray structural analysis of a cLBDH crystal was conducted at 1.58 A. The results obtained show some readily apparent differences around the active sites of cLBDH and BsLBDH. Based on this structural information, a novel (2S, 3S)-BDH having a preferred specificity was developed by introducing a V254L mutation into cLBDH. The influence of this mutation on the stability of cLBDH was not evaluated. Nevertheless, the technique described herein is an effective method for the production of a tailor-made BDH.

To investigate the mechanism for low pH adaptation by a carboxylesterase, structural and biochemical analyses of EstFa_R (a recombinant, slightly acidophilic carboxylesterase from Ferroplasma acidiphilum) and SshEstI (an alkaliphilic carboxylesterase from Sulfolobus shibatae DSM5389) were performed. Although a previous proteomics study by another group showed that the enzyme purified from F. acidiphilum contained an iron atom, EstFa_R did not bind to iron as analyzed by inductively coupled plasma MS and isothermal titration calorimetry. The crystal structures of EstFa_R and SshEstI were determined at 1.6- and 1.5-Å resolutions, respectively. EstFa_R had a catalytic triad with an extended hydrogen bond network that was not observed in SshEstI. Quadruple mutants of both proteins were created to remove or introduce the extended hydrogen bond network. The mutation on EstFa_R enhanced its catalytic efficiency and gave it an alkaline pH optimum, whereas the mutation on SshEstI resulted in opposite effects (i.e. a decrease in the catalytic efficiency and a downward shift in the optimum pH). Our experimental results suggest that the low pH optimum of EstFa_R activity was a result of the unique extended hydrogen bond network in the catalytic triad and the highly negatively charged surface around the active site. The change in the pH optimum of EstFa_R happened simultaneously with a change in the catalytic efficiency, suggesting that the local flexibility of the active site in EstFa_R could be modified by quadruple mutation. These observations may provide a novel strategy to elucidate the low pH adaptation of serine hydrolases.

The crystal structure of a ternary complex of meso-2,3-butanediol dehydrogenase with NAD+ and a competitive inhibitor, mercaptoethanol, has been determined at 1.7 A resolution by means of molecular replacement and refined to a final R-factor of 0.194. The overall structure is similar to those of the other short chain dehydrogenase/reductase enzymes. The NAD+ binding site, and the positions of catalytic residues Ser139, Tyr152, and Lys156 are also conserved. The crystal structure revealed that mercaptoethanol bound specifically to meso-2,3-butanediol dehydrogenase. Two residues around the active site, Gln140 and Gly183, forming hydrogen bonds with the inhibitor, are important but not sufficient for distinguishing stereoisomerism of a chiral substrate.

L(+)-2,3 -Butanediol dehydrogenase (L-BDH) from Brevibacterium saccharolyticum C-1012 has been crystallized by the hanging drop vapor diffusion method with polyethylene glycol 4000 as the precipitant. Crystals of L-BDH belong to the triclinic system, space group P1 with cell dimensions a = 60.8 A, b = 69.2 A, c = 127.4 A , alpha = 96.1 , beta = 100.2 and gama=109.6 . Crystals diffracted to 2.0 A resolution on a synchrotron radiation, and a full data set was collected at resolution of 2.0 A at 100K.c

All oxygenic photosynthetically derived reducing equivalents are utilized by combinations of a single multifuctional electron carrier protein, ferredoxin (Fd), and several Fd-dependent oxidoreductases. We report the first crystal structure of the complex between maize leaf Fd and Fd-NADP(+) oxidoreductase (FNR). The redox centers in the complex--the 2Fe-2S cluster of Fd and flavin adenine dinucleotide (FAD) of FNR--are in close proximity; the shortest distance is 6.0 A. The intermolecular interactions in the complex are mainly electrostatic, occurring through salt bridges, and the interface near the prosthetic groups is hydrophobic. NMR experiments on the complex in solution confirmed the FNR recognition sites on Fd that are identified in the crystal structure. Interestingly, the structures of Fd and FNR in the complex and in the free state differ in several ways. For example, in the active site of FNR, Fd binding induces the formation of a new hydrogen bond between side chains of Glu 312 and Ser 96 of FNR. We propose that this type of molecular communication not only determines the optimal orientation of the two proteins for electron transfer, but also contributes to the modulation of the enzymatic properties of FNR.

2-Dicyanomethylene-1,1,3,4,5,5-hexacyanopentene (DHCP) dianion, which had been reported as 2,3-bis(dicyanomethyl)-1,1,4,4-tetracyanobutadiene, was synthesized as a tetraalkylammonium salt from hexacyanobutadiene. The crystal structures of tetraalkylammonium (alkyl: methyl, ethyl), tetrathiafulvalene (TTF) and bisethylenedithio–TTF (BEDT–TTF or ET) salts were solved. Possible reaction processes to give DHCP were deduced based on its molecular structure. Fourteen solid charge transfer complexes, seven of which were highly conductive, were prepared with mainly TTF derivatives as electron donor. ET afforded two metallic complexes, one of which (ET1) showed a metal–insulator transition at 180 K and the other (ET2) retained metallic behavior down to 1.3 K. BEDO–TTF complex was metallic down to 4.2 K. The crystal, electronic and band structures and EPR parameters of ET1 and ET2 were studied. Both ET1 and ET2 have a β″-type packing mode with significantly different mutual orientation between ET and DHCP molecules. The polymorphism is relevant to both different conformations of DHCP; cis and trans, which demonstrate the flexibility of the molecular shape and conformation, and intermolecular atomic contacts between ET and DHCP molecules.

Although the molecular structure of the cyanocarbon anion of (C14N8)2- had been expected as 2,3-bis(dicyanomethylene)-1,1,4,4-tetracyanobutandiide, the crystal structure analyses revealed that it has the atomic connectivity corresponding to 2-Dicyanomethylene-1,1,3,4,5,5-hexacyanopentenediide (DHCP). This stable dianion is regarded as being consisted of two dicyanomethylene groups and a planar tetracyanobutandiide. Comparing the dihedral angles between these planar parts among the different kinds of the salts, it is concluded that DHCP shows the structural flexibility. This peculiar nature is regarded as the origin to afford the characteristic molecular packing of (TTF)2(DHCP)(CH3CN) in which the segragated stack of TTF and the donor-anion alternating stack co-exist. The salts with BEDT-TTF (ET) and BEDO-TTF (BO) showed the metallic conducting behavior down to liquid helium temperature or below.

The preparation of charge transfer (CT) complexes of dithiooxalate (dto) transition metal salts M(dto)2 (M = Ni, Pd, Pt or Cu) with a variety of electron donor molecules was examined based on the redox properties of constituent molecules. Conventional electron donors of TTF type, such as bis(ethylenedithio)- (BEDT-TTF (ET)) and bis(ethylenedioxy)-tetrathiafulvalene (BEDO-TTF (BO)), mainly afford 2∶1 solid CT complexes composed of a donor cation dimer D+·D+· and dianion of M(dto)2 (A2−) when M = Ni, Pd or Pt. The constituent molecules stack alternately to construct a D+·D+·A2− column. Their crystal structures are unique among the ET and BO complexes so far known, namely the M(dto)22− ions are isolated from each other being surrounded by six dimers of D+· in the crystals. Three kinds of 2∶1 complex were obtained between ET and Pd(dto)2. The electronic spectra of the solids are characterized by the particular intradimer transition of the donor cation dimer. The infrared vibrational spectra strongly exhibit the ag modes of the donor cation due to its dimerization. All of the complexes are electrical insulators. A singlet–triplet magnetic excitation is clearly observed for some of them. A highly conductive 4∶1 complex was obtained between ET and Pd(dto)2, (ET)4[Pd(dto)2], which exhibited electronic absorption extending below 5 × 103 cm−1. The complex shows a semimetallic nature.

A theoretical method to extract structural information on spin-exchange-coupled manganese tetramers from the EPR spectroscopy data is presented. This method has been applied to two EPR data, i.e. the ground-state spin (S = 1/2) and the first excitation energy (30—37 cm−1), for the S2-state Mn tetramer in the photosynthetic oxygen-evolving complex, which exhibits a “g = 2 multiline” EPR signal. Based on the EXAFS data and the manganese chemistry, a simplified model spin Hamiltonian to describe the S2-state Mn cluster will be presented, such that two spin-exchange interactions due to 2.7—2.8 A and/or 3.2—3.5 A Mn–Mn bonds can vary from weak to strong coupling, sensitively, depending upon the bridge structure, except for a strong-antiferromagnetic interaction due to a 2.7 A Mn(III)–Mn(IV) bond and the other weak ones. By computer-search of the possible spin-exchange structures with respect to these two parametric interactions, it was found that (1) a dimer of di-μ2-oxo bridged Mn dimers, a propeller-type tet...

Possible spin-exchange structures of the Mn(III,IV,IV,IV) cluster in an S 2 state of plant photosystem II were computer-searched, within the range compatible with X-ray absorption data, by diagonalizing each Heisenberg spin-exchange Hamiltonian and then by checking whether it can take the S =1/2 ground state capable of explaining the effective 55 Mn hyperfine constants determined from oriented multiline spectra and the first excited state with excitation energy around 20–50 cm −1 , or not. The possible spin-exchange structures were found to be distributed in those that contain only one strong-antiferromagnetic coupling and at most two intermediate coupling(s). The most probable structures are discussed in detail.

Isomaltase from Saccharomyces cerevisiae is an oligo-1,6-glucosidase that preferentially hydrolyzes isomaltose, with little activity towards isomaltotriose or longer oligosaccharides. An amino-acid sequence analysis of the isomaltase revealed that it belongs to glucoside hydrolase family 13. Recombinant isomaltase was purified and crystallized by the hanging-drop vapour-diffusion method with PEG 3350 as the precipitant. The crystals belonged to space group C2, with unit-cell parameters a = 95.67, b = 115.42, c = 61.77 A, beta = 91.17 degrees . X-ray diffraction data were collected to 1.35 A resolution from a single crystal on a synchrotron-radiation source.

Abstract: Crystals of the P-amylase from Bacillus cereus var. mycoides were grown by the vapor diffusion method with polyethylene glycol 6000 as the precipitant. These crystals belong to the monoclinic system, space group C2 with a=177.9 A, b = 112.9 A, c = 146.2 A and /J=105.8 °. They diffracted well to 2.0 A resolution on a synchrotron radiation source, a full data set being collected to 2.2 A resolution. On the basis of the Matthews' parameter (Vm), at least four molecules are estimated to be in the asymmetric unit.

Applying the UDFT/B3LYP/(lacvp**, lacv3p**) geometry optimization method together with a Poisson-Boltsman equation solver in the e = 4 dielectric medium to a version-upped “truncated-OEC-cluster” model of MT-type, we found that (1) Upon the Si-state transitions (I = 0 — 4) in a cyclic change of the most-stable tautomer(s), a proton release pattern of 1:0:1:2 has been derived with use of calculated exothermic vs endothermic energies, to yield the oxidation states: S0Mna III; Mnb III, Mnc III, Mnd IV, S1Mna III; Mnb IV, Mnc III, Mnd IV, S2 +Mna IV; Mnb IV, Mnc III, Mnd IV, S3 +Mna IV; Mnb IV, Mnc IV, Mnd IV and S4aMna IV; Mnb IV, Mnc IV, Mnd IV, (2) The redox potential for the last S3/S4a + oxidation step has been evaluated to be ca.1.07 V, a significantly-reduced value due to the H-bonding network between YZ, H190 and Ca 2+-binding site in the Mn4Ca cluster, (3) The S4a-intermediate contains the catalytic Mna IV ion binding two adjoining substrate derivatives, a hydroxyl anion (W1 = HO−) and an oxo radical (W2 = O−·), and (4) The O-O bond formation is thermally induceable by a proton-coupled electron transfer (PCET) via a transition state with an activation energy of ca. 11.2 kcal/mol and a small exothermicity of ca. −4.5 kcal/mol, to yield a side-on superoxo anion radial bound to the Mna III ion in the second intermediate, formulated as S4bMna III:O2−·(W1 = W2); Mnb IV, Mnc III, Mnd IV, where the third Mnc III ion is in a low-spin state of S c=1.

Studies of the crystal structures and formation of a charge-transfer (CT) complex proved that 2,2′-bi-1H-imidazole (H2BIM0) forms a new system concerned with the interplay of proton-transfer (PT) and electron-transfer (or charge-transfer (CT)) interactions. X-Ray crystal analyses of newly isolated species, 2-(2-1H-imidazolyl)-1H-imidazolium (H3BIM+) and 2,2′-bi-1H-imidazolium (H4BIM2+), revealed the formation of strong hydrogen bonds along the side-by-side direction of the molecular plane. CT complexes of H3BIM+ and H4BIM2+ with the 7,7,8,8-tetracyanoquinodimethane derivatives (TCNQs) were synthesized by a method metathesis, and the crystal structure of (H3BIM+)2(TCNQ−)2(TCNQ0) was determined. During CT complex formation, the initial simple protonated states of [H3BIM+] or [H4BIM2+] changed to different ones, for example, [H4BIM2+]x[H3BIM+]1−x, [H3BIM+]x[H2BIM0]1−x, and H2BIM0. According to the protonated states of the H2BIM system, the formal charge of the TCNQs in the CT complexes varies from completely...

2-alkyloxy substituted tetracyanoallyl anions (RO-TCA−, R = Me, Et, Pr, Bu) were examined in the formation of the radical cation salts of organic donor molecules systematically. 14 kinds of salts have been obtained so far. Among them, (BEDO-TTF)2(EtO-TCA)(H2O)0.75 showed weak metallic temperature dependence of the conductivity down to ca. 180 K ([sgrave]rt = 30 S cm−1). The crystal structure analyses of (BEDT-TTF)2(PrO-TCA) (needle form) and (TTT)(MeO-TCA) revealed the layered structure in both cases. The two C(CN)2 groups in an anion are twisted to each other in either salts. The structures of the anion layers showed the existence of various packing patterns for this anion series.

The characters of hydrogen-bond of 2,2′-bi-1H-imidazole (H2BIM) system were examined from a viewpoint of the proton-transfer (PT) and electron-transfer (CT) interactions. To make the effects of the dibenzo substitution of H2BIM molecule clear, we prepared the charge-transfer complexes of dibenzo-2,2′-bi-1H-imidazole (H2BBIM) and H2BIM systems with 7,7,8,8- tetracyanoquinodimethane(TCNQ). The one-dimensional column of TCNQ was confirmed by the crystal structural analysis, however, the packing forms of the H2BIM and H2BBIM systems were entirely different to each other. The H2BBIM system in the TCNQ complex formed the one-dimensional column which is parallel to the TCNQ stack, while the H2BIM system was constructed by the hydrogen-bonded dimer unit along the perpendicular direction to the TCNQ column.

The complex formation of bis(ethylenedioxy)tetrathiafulvalene (BEDO-TTF) with 29 organic electron acceptors and six organic anions yielded 37 charge-transfer (CT) complexes, about three-quarters of which exhibited metallic behavior. The BEDO-TTF molecule proves to be an excellent source for stable metals irrespective of the structure, shape, size, and electron affinity of counter components. The crystal structure of metallic BEDO-TTF complexes indicates a strong aggregation of donor molecules into a two-dimensional (2D) layered structure by the aid of both intermolecular CH···O and side-by-side heteroatom contacts. Band calculations based on an extended Huckel method demonstrate that the sulfur atoms of the BEDO-TTF molecule are dominant for 2D intermolecular overlap. The resulting 2D electronic bands with large band width serve to stabilize the metallic state even at low temperatures. A comparison between the structural properties of complexes of BEDO-TTF and those of its sulfur analog BEDT-TTF (BEDT-TTF...

No crystal structures are yet available for homologues of a predicted acetamidase/formamidase (Amds/Fmds) from the archaeon Thermoanaerobacter tengcongensis. The Amds/Fmds gene was cloned and expressed as a soluble protein in Escherichia coli. Native Amds/Fmds and its SeMet-substituted form were purified and crystallized by vapour diffusion in hanging drops at 296 K. The native crystals, which were grown in PEG 8000, belong to the monoclinic space group P2(1), with unit-cell parameters a = 41.23 (3), b = 152.88 (6), c = 100.26 (7) A, beta = 99.49 (3) degrees. The diffraction data were collected to 2.00 A resolution using synchrotron radiation. Based on a predicted solvent content of 50%, a Matthews coefficient of 2.44 A3 Da(-1) and two main peaks in the self-rotation function, the asymmetric unit is predicted to contain two dimers of the 32 kDa native protein. MAD data were collected for the SeMet protein, but the corresponding crystals display different unit-cell parameters and appear to contain four dimers in the asymmetric unit.

Trifluoroacetaldehyde azine (TFAcAz) was prepared and its reactivity was investigated. TFAcAz is readily attacked by nucleophiles such as trialkylamines, methanol, and water. The structures of the products were studied by 1H, 13C, and 19F NMR, IR, Raman, and EI–mass spectroscopies and X–ray crystallographic analysis. A cyclic dimer containing a 1,4-dihydro-1,2,4,5-tetrazine ring was obtained by the reactions of TFAcAz with trialkylamines. 1,2-Adducts were found to be produced by the reactions of TFAcAz with methanol and with water.

A new type of 90–240 G wide EPR signal from the modified S3 state of Ca2+-depleted photosystem II (PSII) is concluded to arise from a partially oxidized water radical with spin S = 1 2 interacting with the S = 1 2 S2-state manganese tetramer (‘Mn4’). This is based exclusively on the fact that the average g value of the radical is ≈ 2.010–2.012, a value close to that of OH radical (2.011) and significantly larger than either one of an oxidized imidazole (2.00226) or an oxidized tyrosine (2.0046), indicating that the radical may be (HOOH)−, the most probable intermediate produced by abstracting two protons and one electron from a bound water dimer. The effective interactions between the Mn4 and radical spins (S1 and S2, respectively) of the form H int = J 12 S 1 · S 2 + S 1 · D 12 · S 12 have been thoroughly investigated to find which Mn4-radical complex can reasonably make both J 12 and D 12 as small as ≈ 100 G in magnitude and can, simultaneously, yield an X-ray absorption Mn K-edge energy 0.7 ± 0.3 eV higher than that in the modified S2 state. As the most probable model, we propose that the radical must form the third bridging ligand between di-μ-oxo or μ2-oxo-(μ3-oxo) bridged Mna(III) and Mnb(IV) ions on the opposite side of mono-μ2-oxo bridged Mnc and Mnd ions.

The electron-accepting ability of a C 60 molecule in solution was evaluated by the measurements of the charge transfer (CT) absorption energy and the redox potential. It was concluded that C 60 is a weak electron acceptor comparable to 2,5-dibromo- p -xyloquinone, s -tetracyanobenzene or 2,3-dichloro-1,4-naphthoquinone. The adibatic electron affinity of a C 60 molecule was estimated as (2.10–2.21)±0.1 eV. Several black solid CT complexes of C 60 were prepared with mainly TTF derivatives and the ionicity of the ground state was examined by their CT band energies and vibrational spectra. These CT complexes were found to be neutral. A crystal structure of the OMTTF complex from benzene was determined.

In order to study the bioactive sites of the glycoprotein hormones, we have prepared five point mutants on the CMGCC (Cys 28 -Met 29 -Gly 30 -Cys 31 -Cys 32 ) region of the human α-subunit by using site-directed mutagenesis. Each mutant human chorionic gonadotropin (hCG) agr; cDNA and a wild-type hCGβ cDNA were transcribed by T3 RNA polymerase, and the mixture of the hCGα mRNA and hCGβ mRNA was microinjected into Xenopus laevis oocytes. All five mutant hCGs produced in oocyte culture supernatants were detected as immunoreactive forms by enzyme immunoassay. In contrast, four mutants (Cys 28 → Tyr 28 , Gly 30 → Arg 30 , Ala 30 , Asp 30 ) were devoid of biological activity in vitro bioassay using the production of testosterone with mouse Leydig cells. These results indicate that the CMGCC region in the α-subunit, particularly the cysteine residue at position 28 and the glycine residue at position 30, plays an important role in the biosynthesis of glycoprotein hormones.