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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.

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 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.

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.

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.

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.

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.

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.

CEL-IV is a C-type lectin isolated from a sea cucumber, Cucumaria echinata. This lectin is composed of four identical C-type carbohydrate-recognition domains (CRDs). X-ray crystallographic analysis of CEL-IV revealed that its tetrameric structure was stabilized by multiple interchain disulfide bonds among the subunits. Although CEL-IV has the EPN motif in its carbohydrate-binding sites, which is known to be characteristic of mannose binding C-type CRDs, it showed preferential binding of galactose and N-acetylgalactosamine. Structural analyses of CEL-IV-melibiose and CEL-IV-raffinose complexes revealed that their galactose residues were recognized in an inverted orientation compared with mannose binding C-type CRDs containing the EPN motif, by the aid of a stacking interaction with the side chain of Trp-79. Changes in the environment of Trp-79 induced by binding to galactose were detected by changes in the intrinsic fluorescence and UV absorption spectra of WT CEL-IV and its site-directed mutants. The binding specificity of CEL-IV toward complex oligosaccharides was analyzed by frontal affinity chromatography using various pyridylamino sugars, and the results indicate preferential binding to oligosaccharides containing Galβ1-3/4(Fucα1-3/4)GlcNAc structures. These findings suggest that the specificity for oligosaccharides may be largely affected by interactions with amino acid residues in the binding site other than those determining the monosaccharide specificity., The Journal of Biological Chemistry, 286(12), pp.10305-10315; 2011

The structure of ferricytochrome c' extracted from Rhodospirillum rubrum has been determined by the X-ray crystallographic method. Crystals in hexagonal space group P6(1), with unit-cell dimensions a = b = 51.72 A and c = 155.49 A, contain one dimer molecule composed of chemically identical polypeptide chains (monomer I and monomer II) per asymmetric unit. An electron density map has been calculated at a resolution of 2.8 A by the multiple isomorphous replacement method using four-circle diffractometer data from native crystals and two heavy-atom derivatives. The quality of the map was improved by averaging the electron density about the non-crystallographic 2-fold axis relating the two monomers. The initial three-dimensional model of monomer I was built on a computer graphics system and that of monomer II was derived from monomer I using the non-crystallographic symmetry matrices. The dimer structure has been refined using a combination of simulated annealing and conventional restrained least-squares crystallographic refinement. The current model includes 244 amino acid residues (122 x 2) and 2 hemes, with a root-mean-square deviation in bond lengths from ideal values of 0.022 A. The current crystallographic R-factor is 23.3% for 4,481 independent reflections [magnitude of Fo greater than or equal to sigma (F)] between 5.0 and 2.8 A resolution. The monomer molecule is structurally organized as an array of four nearly parallel alpha-helices which construct a left-twisted bundle. One end of the bundle, in which a covalently bound protoheme IX prosthetic group is incorporated, is more divergent than the other.(ABSTRACT TRUNCATED AT 250 WORDS)

The three-dimensional structure of Cu,Zn-superoxide dismutase from spinach leaves has been determined by X-ray crystal structure analysis. The atomic coordinates were refined at 2.0 A resolution using the Hendrickson and Konnert program for stereochemically restrained refinement against structure factors, which allowed the use of non-crystallographic symmetry. The crystallographic residual error for the refined model was 24.9%, with a root mean square deviation of 0.03 A from the ideal bond length and an average atomic temperature factor of 9.6 A. A dimeric molecule of the enzyme is comprised of two identical subunits related by a non-crystallographic 2-fold axis. Each subunit of 154 amino acid residues is composed primarily of eight anti-parallel beta-strands that form a flattened cylinder, plus three external loops. The main-chain hydrogen bonds primarily link the beta-strands. The overall structure of this enzyme is quite similar to that of the bovine dismutase except for some parts. The single disulfide bridge (Cys57-Cys146) and the salt bridge (Arg79-Asp101) may stabilize the loop regions of the structure. The Cu2+ and Zn2+ ions in the active site lie 6.1 A apart at the bottom of the long channel. The Cu2+ ligands (ND1 of His-46, and NE2 of His-48, -63, and -120) show an uneven tetrahedral distortion from a square plane. The Zn2+ ligands (ND1 of His-63, -71, and -80 and OD1 of Asp-83) show an almost tetrahedral geometry. The imidazole ring of His-63 forms a bridge between the Cu2+ and Zn2+ ions.(ABSTRACT TRUNCATED AT 250 WORDS)

L-carnosine is a bioactive dipeptide (beta-alanyl-L-histidine) present in mammalian tissues, including the central nervous system, and has potential neuroprotective and neurotransmitter functions. In mammals, two types of L-carnosine-hydrolyzing enzymes (CN1 and CN2) have been cloned thus far, and they have been classified as metallopeptidases of the M20 family. The enzymatic activity of CN2 requires Mn(2+), and CN2 is inhibited by a nonhydrolyzable substrate analog, bestatin. Here, we present the crystal structures of mouse CN2 complexed with bestatin together with Zn(2+) at a resolution of 1.7 A and that with Mn(2+) at 2.3 A CN2 is a homodimer in a noncrystallographic asymmetric unit, and the Mn(2+) and Zn(2+) complexes closely resemble each other in the overall structure. Each subunit is composed of two domains: domain A, which is complexed with bestatin and two metal ions, and domain B, which provides the major interface for dimer formation. The bestatin molecule bound to domain A interacts with several residues of domain B of the other subunit, and these interactions are likely to be essential for enzyme activity. Since the bestatin molecule is not accessible to the bulk water, substrate binding would require conformational flexibility between domains A and B. The active site structure and substrate-binding model provide a structural basis for the enzymatic activity and substrate specificity of CN2 and related enzymes.

High-resolution XANES (X-ray Absorption Near Edge Structure) spectroscopy for Mn in the S1 and S2 states of the spinach photosynthetic O2-evolving complex revealed distinct features in K-edge spectra, when a high signal-to-noise (S/N) ratio of ca. 80 with a low and constant background-to-signal (B/S) ratio of 0.15 to 0.18 was attained. Six features resolved in each S-state spectrum involve a pre-edge feature due to 1s----3d transitions, a main-edge feature possibly due to 1s----4s transitions and four fine structures superimposed on the principal absorption bands due to 1s----4p* transitions. The high-quality pre-edge features were analyzed according to a parametric ligand-field theory in comparison with those of some typical authentic Mn complexes. It was deduced that i) all of the four Mn ions in the S1-state are octahedrally coordinated and two of them constitute a di-mu-oxo bridged Mn(III, III) dimeric subunit; ii) the bridged Mn(III) ions are further bridged by a deprotonated water dimer, (HOHOH)-, and coordinated by imidazole-N and carboxylate-O- on the opposite side of the Mn atom from the di-mu-oxo bridge; iii) the other two Mn ions exist in the form of Mn(III) monomeric subunits; and iv) upon the S1----S2 transition, only the bridged Mn(III,III) is oxidized to Mn(III,IV). The distinct change in the principal absorption band shape upon the S1----S2 transition is briefly discussed to obtain the XANES evidence for a tetrameric Mn-cluster.

CEL-III is a Ca(2+)-dependent hemolytic lectin, isolated from the marine invertebrate Cucumaria echinata. The three-dimensional structure of CEL-III/GalNAc and CEL-III/methyl alpha-galactoside complexes was solved by x-ray crystallographic analysis. In these complexes, five carbohydrate molecules were found to be bound to two carbohydrate-binding domains (domains 1 and 2) located in the N-terminal 2/3 portion of the polypeptide and that contained beta-trefoil folds similar to ricin B-chain. The 3-OH and 4-OH of bound carbohydrate molecules were coordinated with Ca(2+) located at the subdomains 1alpha, 1gamma, 2alpha, 2beta, and 2gamma, simultaneously forming hydrogen bond networks with nearby amino acid side chains, which is similar to carbohydrate binding in C-type lectins. The binding of carbohydrates was further stabilized by aromatic amino acid residues, such as tyrosine and tryptophan, through a stacking interaction with the hydrophobic face of carbohydrates. The importance of amino acid residues in the carbohydrate-binding sites was confirmed by the mutational analyses. The orientation of bound GalNAc and methyl alpha-galactoside was similar to the galactose moiety of lactose bound to the carbohydrate-binding site of the ricin B-chain, although the ricin B-chain does not require Ca(2+) ions for carbohydrate binding. The binding of the carbohydrates induced local structural changes in carbohydrate-binding sites in subdomains 2alpha and 2beta. Binding of GalNAc also induced a slight change in the main chain structure of domain 3, which could be related to the conformational change upon binding of specific carbohydrates to induce oligomerization of the protein.

The body fluid of marine invertebrate Cucumaria echinata (Holothuroidea) contains four Ca2+-dependent galactose-specific lectins. One of these lectins, CEL-IV, is composed of a C-type carbohydrate-recognition domain homotetramer. CEL-IV exhibits higher specificity for α-galactosides than for β-galactosides, while other C. echinata lectins show preferential binding of β-galactosides. We constructed an artificial synthetic gene for recombinant CEL-IV (rCEL-IV) based on the amino acid sequence previously determined from the purified protein. rCEL-IV was expressed in Escherichia coli cells as inclusion bodies. After the refolding process, most of rCEL-IV spontaneously formed a homotetramer structure having interchain disulfide bonds. The secondary structure of rCEL-IV was similar to that of the native one, as judged by the comparison of the far UV-circular dichroism spectra of rCEL-IV and native CEL-IV (nCEL-IV). Carbohydrate-binding specificity of rCEL-IV was confirmed to be similar to that of nCEL-IV from the results of the binding-inhibition assay using liposomes composed of rabbit erythrocyte lipids. Crystals of rCEL-IV were obtained in a few days by the sitting drop vapor diffusion method. These results indicate that rCEL-IV achieved essentially correct three-dimensional structure, including the carbohydrate-binding sites, and it would be very useful for further study on the carbohydrate-recognition mechanism by mutational and X-ray crystallographic analyses.

The crystal structures of beta-amylase from Bacillus cereus var. mycoides in complexes with five inhibitors were solved. The inhibitors used were three substrate analogs, i.e. glucose, maltose (product), and a synthesized compound, O-alpha-D-glucopyranosyl-(1-->4)-O-alpha-D-glucopyranosyl-(1-->4)-D-xylopyranose (GGX), and two affinity-labeling reagents with an epoxy alkyl group at the reducing end of glucose. For all inhibitors, one molecule was bound at the active site cleft and the non-reducing end glucose of the four inhibitors except GGX was located at subsite 1, accompanied by a large conformational change of the flexible loop (residues 93-97), which covered the bound inhibitor. In addition, another molecule of maltose or GGX was bound about 30 A away from the active site. A large movement of residues 330 and 331 around subsite 3 was also observed upon the binding of GGX at subsites 3 to 5. Two affinity-labeling reagents, alpha-EPG and alpha-EBG, were covalently bound to a catalytic residue (Glu-172). A substrate recognition mechanism for the beta-amylase was discussed based on the modes of binding of these inhibitors in the active site cleft.

The crystal structure of glucose dehydrogenase (GlcDH) from Bacillus megaterium IWG3 has been determined to an R-factor of 17.9% at 1.7 A resolution. The enzyme consists of four identical subunits, which are similar to those of other short-chain reductases/dehydrogenases (SDRs) in their overall folding and subunit architecture, although cofactor binding sites and subunit interactions differ. Whereas a pair of basic residues is well conserved among NADP(+)-preferring SDRs, only Arg39 was found around the adenine ribose moiety of GlcDH. This suggests that one basic amino acid is enough to determine the coenzyme specificity. The four subunits are interrelated by three mutually perpendicular diad axes (P, Q, and R). While subunit interactions through the P-axis for GlcDH are not so different from those of the other SDRs, those through the Q-axis differ significantly. GlcDH was found to have weaker hydrophobic interactions in the Q-interface. Moreover, GlcDH lacks the salt bridge that stabilizes the subunit interaction in the Q-interface in the other SDRs. Hydrogen bonds between Q-axis related subunits are also less common than in the other SDRs. The GlcDH tetramer dissociates into inactive monomers at pH 9.0, which can be attributed mainly to the weakness of the Q-axis interface.

The crystal structure of beta-amylase from Bacillus cereus var. mycoides was determined by the multiple isomorphous replacement method. The structure was refined to a final R-factor of 0.186 for 102,807 independent reflections with F/sigma(F) > or = 2.0 at 2.2 A resolution with root-mean-square deviations from ideality in bond lengths, and bond angles of 0.014 A and 3.00 degrees, respectively. The asymmetric unit comprises four molecules exhibiting a dimer-of-dimers structure. The enzyme, however, acts as a monomer in solution. The beta-amylase molecule folds into three domains; the first one is the N-terminal catalytic domain with a (beta/alpha)8 barrel, the second one is the excursion part from the first one, and the third one is the C-terminal domain with two almost anti-parallel beta-sheets. The active site cleft, including two putative catalytic residues (Glu172 and Glu367), is located on the carboxyl side of the central beta-sheet in the (beta/alpha)8 barrel, as in most amylases. The active site structure of the enzyme resembles that of soybean beta-amylase with slight differences. One calcium ion is bound per molecule far from the active site. The C-terminal domain has a fold similar to the raw starch binding domains of cyclodextrin glycosyltransferase and glucoamylase.

The cDNA of the common alpha-subunit of human glycoprotein hormone was mutated by site directed mutagenesis in the CMGCC region composed of cysteine-methionine-glycine-cysteine-cysteine (position 28-32). The cDNA of wild-type human thyrotropin (hTSH) beta-subunit and that of wild-type or mutant common alpha-subunits were co-transfected into COS-I cells. The concentration of hTSH determined by two immunoradiometric assay systems was detectable in culture media of COS-I cells transfected with wild-type (CMGCC) and a mutant (CRGCC) alpha-subunits but not four other mutants (YMGCC) (CMRCC) (CMACC) (CMDCC). The present data with the other studies on wild-type or mutant glycoprotein hormones support our hypothesis that an amino acid motif of "C-X-G-X-C" in the common alpha-(CMGCC in human) and beta-(CAGYC in human) subunits play an important role in biosynthesis of glycoprotein hormones in all species.

Bioactive peptides of different sources and biological functionalities, like endothelins, sarafotoxins, bee and scorpion venom toxins, contain a consensus cystine framework, Cys-(X)1-Cys/Cys-(X)3-Cys, which has been found to induce and stabilize a homologous folding motif named the cystine-stabilized alpha-helix (CSH). This is composed of an alpha-helical segment spanning the Cys-(X)3-Cys sequence portion that is crosslinked by two disulfide bridges to the sequence portion Cys-(X)1-Cys, itself folded in an extended beta-strand type structure. Search for sequence homologies of peptides and proteins in the SWISS-PROT and PDB data banks provided additional multiple examples of this type of cystine framework in serine proteinase inhibitors, in insect and plant defense proteins, as well as in members of the growth factor family with the cystine-knot. A comparative analysis of the known 3D-structures of these peptides and proteins confirmed that the presence of this peculiar cystine framework leads in all cases to a high degree of local structural homology that consists of the CSH motif, except for the cystine-knot, of the superfamily of the growth factors. In this case the cyclic structure formed by the parallel cysteine connectivities of Cys-(X)1-Cys/Cys-(X)3-Cys framework is penetrated by a third disulfide bond with formation of a concatenated knot, and the two disulfide-bridged peptide chains Cys-(X)1-Cys and Cys-(X)3-Cys are located in beta-strands. Conversely, peptides and proteins containing Cys-(X)m-Cys/Cys-(X)n-Cys cystine frameworks that differ from m/n = 1/3 were found to fold only sporadically into local alpha-helical structures.

Flash-induced changes of the Mn K-edge absorption spectra have been studied in the oxygen-evolving complex depleted of Ca. The Mn K-edge energy for the Ca-depleted S1-state was lower by 1.5 eV than that for the normal S1-state. The K-edge energy upshifted by 1 eV after one flash, indicative of an oxidation of Mn. After two flashes, the K-edge was elevated as well by 0.4 eV, and then reached a steady-state high level after continuous illumination where the K-edge energy was higher by 0.9 eV than that after one flash. The results indicate that the Mn-cluster and/or its direct ligand could be oxidized up to two electrons but further events are blocked.

Crystals of a [2Fe-2S]ferredoxin (Fd) I with a relative molecular mass of 10,480 were obtained from the blue-green alga Aphanothece sacrum . Each asymmetric unit of the crystal contains four molecules. An electron density map calculated by the single isomorphous replacement method with the anomalous dispersion at 2·5 A resolution was refined by averaging the four molecules in the asymmetric unit. Positional and isotropic thermal parameters for the non-hydrogen atoms of the four molecules and 158 water molecules were refined to an R -factor ( R = Σ| F o − F c |/Σ F o ) of 0·23 by the restrained least-squares method. The estimated root-mean-square (r.m.s.) error for the atomic positions is 0·3 A. The r.m.s. deviations of equivalent C α atoms of the asymmetric-unit molecules superposed by the least-squares method average 0·35 A. The Fd molecule has a structure like the β-barrel in the molecule of the [2Fe-2S] Fd from Spirulina platensis . A [2Fe-2S] cluster is bonded covalently to the protein molecule by four Fe-S, in which three of the Fe-S bonds are in a loop segment from position 38 to 47. The hydrophobic core inside the β-barrel is formed by seven conservative residues: Val15, Val18, Ile24, Leu51, Ile74, Ala79 and Ile87. The molecular surface around Tyr23, Tyr80 and the active center may interact with ferredoxin-NADP + reductase. One of the two iron atoms of the [2Fe-2S] cluster should be more easily reduced than the other because of differences in the hydrogen-bonding scheme and the hydrophobicity around the atoms.

The structure of tetraheme cytochrome c3 isolated from Desulfovibrio vulgaris Miyazaki has been determined at 2.5 A resolution by an X-ray diffraction method. Protein phases were computed by the multiple isomorphous replacement method using the native and four heavy atom derivatives, anomalous scattering measurements of the latter being considered. The mean figure of merit was 0.77. Four heme groups are exposed on the surface of the molecule. There are some short helical segments in the polypeptide chain, and hair-pin turns are often observed at glycine and alanine residues.

The structure of cytochrome c3 from Desulfovibrio vulgaris Miyazaki (DvM) is considered in detail by scrutinizing main chain folding together with the structure of the protein from D. desulfuricans Norway (DdN). The relative arrangement of the four heme groups in this molecule is similar to that of DdN and the disposition of alpha-carbon atoms of cysteine and histidine residues binding to heme groups is also similar. Structural differences between the two proteins occur in the shape of some specific loops of the chain on the molecular surface. As a result of a careful comparison of structures and sequences in both cytochromes c3, the reported sequence alignment of the cytochrome c3 family has been revised. Our new proposal of a sequence alignment based on the three-dimensional structures contains 24 evolutionarily conservative residues. All these conservative residues may play an important role in the folding pattern of cytochromes c3.

The crystal structure of Taka-amylase A was studied by an X-ray diffraction method at 3 A resolution. A total of 452 amino acid residues were found from the electron density map at the present stage. The four disulfide bonds and the branched carbohydrate were also located on the map. The difference electron density map of the maltotriose-soaked crystal showed that a maltose unit was bound in the active center left. The binding of iodine atoms to the enzyme was also studied.

The molecular structure of Taka-amylase A, an alpha-amylase from Aspergillus oryzae, has been studied at 6 A resolution by X-ray diffraction analysis. The electron density map showed a non-crystallographic three-fold screw arrangement of the molecules in the crystal. The molecule is an ellipsoid with approximate dimensions of 80 x 45 x 35 A and contains a hollow which may correspond to the active center. The inhibitor molecules bind to Taka-amylase A at four different sites, one of which is located in the hollow of the enzyme. The probable position of a thiol group is discussed in connection with heavy atom binding.

A complete molecular model of Taka-amylase A consisting of 478 amino acid residues was built with the aid of amino acid sequence data. Some typical structural features of the molecule are described. A model fitting of an amylose chain in the catalytic site of the enzyme showed a possible productive binding mode between substrate and enzyme. On the basis of the difference Fourier analysis and the model fitting study, glutamic acid (Glu230) and aspartic acid (Asp297), which are located at the bottom of the cleft, were concluded to be the catalytic residues, serving as the general acid and base, respectively.

In order to elucidate the importance of a ferredoxin (Fd) Arg-Glu pair involved in dynamic exchange from intra- to intermolecular salt bridges upon complex formation with ferredoxin-NADP+ oxidoreductase (FNR), Equisetum arvense FdI and FdII were investigated as normal and the pair-lacking Fd, respectively. The FdI mutant lacking this pair was unstable and rapidly lost the [2Fe–2S] cluster. The catalytic constant (kcat) of the electron transfer for FdI is 5.5 times that for FdII and the introduction of this pair into FdII resulted in the increase of kcat to a level comparable to that for FdI, demonstrating directly that the Arg-Glu pair is important for efficient electron transfer between Fd and FNR.

2,3-Butanediol dehydrogenase (BDH) catalyzes the NAD-dependent redox reaction between acetoin and 2,3-butanediol. There are three types of homologous BDH, each stereospecific for both substrate and product. To establish how these homologous enzymes possess differential stereospecificities, we determined the crystal structure of l -BDH with a bound inhibitor at 2.0 A. Comparison with the inhibitor binding mode of meso-BDH highlights the role of a hydrogen-bond from a conserved Trp residue192. Site-directed mutagenesis of three active site residues of meso-BDH, including Trp190, which corresponds to Trp192 of l -BDH, converted its stereospecificity to that of l -BDH. This result confirms the importance of conserved residues in modifying the stereospecificity of homologous enzymes.

The Fab fragment of a monoclonal anti-idiotopic antibody against M315 has been isolated and its complexes with Fv and Fab′ fragment of M315 have been crystallized by using poly(ethylene glycol) 6000 or ammonium sulfate. X-ray diffraction photographs showed that the crystal of the complex with Fv diffracts better than that with Fab′. The Fv-complexed crystal was shown to be tetragonal I 4, with cell dimensions α = 152 A and c = 69 A , and to contain one complex molecule of about 75000 molecular weight in the crystallographic asymmetric unit.

The crystal structure of aspartate aminotransferase of Escherichia coli was determined by X-ray structure analysis at 2.8 A resolution. The structure was solved by the molecular replacement method and refined to an R-factor of 0.27, and it was found that the overall structure of AspAT of E. coli is similar to that of those of higher animals.

Cytochrome c553 from the sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki, has been crystallized. The combination of microdialysis and vapor diffusion allowed successful crystallization. The crystals were of good quality, and useful data were obtained that extended to the nominal resolution of 1.3 A. The space group is P4(3)2(1)2 with cell dimensions of a = b = 42.7 A, c = 103.4 A. More than twenty heavy-atom reagents were screened with the isomorphous replacement technique, and only the mersalyl derivative could be used for the phase determination. The single isomorphous replacement method combined with the anomalous scattering effect of the Hg-atom in mersalyl and the Fe-atom of the heme group was used for the phase determination.

The structure of cytochrome c3 from the sulfate-reducing bacterium Desulfovibrio vulgaris Miyazaki has been successfully refined at 1.8 A resolution. The crystallographic R factor is 0.176 for 9907 significant reflections. The isotropic temperature factors of individual atoms were refined and a total of 47 water molecules located on the difference map were incorporated in the refinement. The four heme groups are closely packed, with adjacent pairs of heme planes being nearly perpendicular to each other. The fifth and the sixth ligands of the heme iron atoms are histidine residues with N epsilon 2-Fe distances ranging from 1.88 A to 2.12 A. The histidine co-ordination to the heme iron is different for each heme group. The heme groups are all highly exposed to solvent, although the actual regions exposed differ among the hemes. The four heme groups are located in different environments, and the heme planes are deformed from planarity. The differences in the heme structures and their environments indicate that the four heme groups are non-equivalent. The chemical as well as the physical properties of cytochrome c3 should be interpreted in terms of the structural non-equivalence of the heme groups. The characteristic secondary structural non-equivalence of the heme groups. The characteristic secondary structures of the polypeptide chain of this molecule are three short alpha-helices, two short beta-strands and ten reverse turns.

The aspartate aminotransferase of Escherichia coli was overproduced in cells after genetic manipulation, and was crystallized from a polyethylene glycol solution, pH 7.0. The crystals obtained were of good quality and had diffractions extending beyond 2.4 A. The space group and unit cell dimensions were determined with a precession camera and a four-circle diffractometer to be C222(1), and a = 157.1 A, b = 85.5 A, and c = 79.7 A, respectively. Only one protein subunit is contained in an asymmetric unit.

A lectin from Flammulina veltipes (Enoki-dake) has been crystallized in a form suitable for crystallographic structure analysis. Two types of crystals were grown: one from polyethylene glycol 6000 solution at pH 7 and the other from ammonium sulfate solution at pH 7. The latter type is more suitable for the crystallographic investigations because of its high resolution X-ray diffraction and smaller number of asymmetric molecules.