Inhibition of class II major histocompatibility complex antigen processing by Escherichia coli heat-labile enterotoxin requires an enzymatically active A subunit

Escherichia coli heat-labile enterotoxin (LT) and cholera toxin (CT) were found to inhibit intracellular antigen processing. Processing was not inhibited by mutant LT with attenuated ADP-ribosyltransferase activity, CT B or LT B subunit, which enhanced presentation of preexisting cell surface peptide-class II major histocompatibility complex complexes. Inhibition of antigen processing correlated with A subunit ADP-ribosyltransferase activity.     

Inhibition of Escherichia coli heat-labile enterotoxin by an amino carbonyl product, lactose-alpha-lactalbumin

The inhibition by lactose-alpha-lactalbumin amino carbonyl product of Escherichia coli heat-labile enterotoxin was studied by GM1-ELISA and by assay with CHO-K1 cells. The product dose-dependently inhibited the binding of the enterotoxin to GM1 ganglioside and decreased the morphological change of CHO-K1 cells caused by this toxin. The results suggest that this product may be a receptor analogue in the intestine.     

In vitro effects of a high-molecular-weight heat-labile enterotoxin from enteroaggregative Escherichia coli

The pathogenic mechanisms of enteroaggregative Escherichia coli (EAggEC) infection are not fully elucidated. In this work we show that an ammonium sulfate precipitate of culture supernatant of EAggEC strain 049766 increased the potential difference (PD) and the short-circuit current (Isc) in rat jejunal preparations mounted in Ussing chambers. The precipitate contained two major proteins of 108 and 116 kDa, which were partially copurified by chromatography in DEAE-cellulose. This chromatographic fraction (peak I) increased jejunal PD and Isc in a dose-dependent manner, accompanied by a decrease in tissue electrical resistance. These effects were inhibited by incubation of peak I at 75 degreesC for 15 min or for 1 h with proteinase K at 37 degreesC. Rabbit polyclonal antibodies against peak I containing both the 108- and 116-kDa proteins inhibited the enterotoxic effect. Specific polyclonal antibodies raised against the 108-kDa but not against the 116-kDa protein inhibited the enterotoxic effect, suggesting that the 108-kDa protein is the active toxic species. Moreover, another EAggEC strain (065126) producing the 116-kDa protein but not the 108-kDa protein had no effect on rat jejunal mucosa in the Ussing chamber. The >100-kDa fraction derived from prototype EAggEC strain 042, which also expressed both 108- and 116-kDa proteins, also produced an enterotoxic effect on rat jejunal preparations in Ussing chambers; however, the same strain cured of its 65-MDa adherence plasmid did not. A subclone derived from the 65-MDa plasmid expressing the 108-kDa toxin (and not the 116-kDa protein) elicited rises in Isc. Tissue exposed to any preparation containing the 108-kDa toxin exhibited similar histopathologic changes, characterized by increased mucus release, exfoliation of cells, and development of crypt abscesses. Our data suggest that some EAggEC strains produce a ca. 108-kDa enterotoxin/cytotoxin which is encoded on the large virulence plasmid.     

A chimeric inorganic pyrophosphatase derived from Escherichia coli and Thermus thermophilus has an increased thermostability

Factors contributing to the thermostability of inorganic pyrophosphatase (PPase) were investigated by examining chimeric PPases from Escherichia coli and Thermus thermophilus (Tth). Two chimeric PPase genes, T1-135E (residues 1-135 from the N terminus are comprised of Tth PPase and residues 136-173 are derived from the C terminus of E. coli PPase) and T1-149E [residues 1-149 from the N terminus are from Tth PPase and the rest (150-175) are from E. coli PPase], were constructed by random chimeragenesis. After the genes were overexpressed in the E. coli BL21(DE3) strain and the expression products were purified, we compared the characteristics of these chimeric PPases with those of the parental PPases. We found that the two chimeras had higher activity than either parent PPase at the optimum temperature. We also examined thermal stability in terms of CD spectra, fluorescence spectra, and thermal changes in enzyme activity. The results revealed that the thermal stability of T1-149E is similar to that of Tth PPase, but T1-135E is much more stable. This suggests that the four residues that are different between T1-135E and T1-149E may be critical for thermostability between the two chimeras. By comparing the three-dimensional structures of Tth and E. coli PPases, we deduced that the following two factors may contribute to differences in thermostability. (1) Two residues (Thr138 and Ala141 in the Tth PPase and His140 and Asp143 in the E. coli PPase) in the vicinity of the trimer-trimer interface were different. (2) The Ala144-Lys145 loop in the Tth PPase was deleted in the E. coli PPase and also in the T1-135E chimera. Therefore, we conclude that T1-135E was thermostabilized by these two factors, and also, the Tth PPase moiety may contribute to the structural integrity of the chimeric enzymes.     

Display of an inhibin epitope in a surface-exposed loop of the E. coli heat-labile enterotoxin B subunit

In vitro gene manipulation was used to develop a novel chimeric antigen consisting of the non-toxic B subunit (EtxB) of an E. coli enterotoxin and the first 14 N-terminal amino acid residues of the carboxy-terminal portion of the alpha subunit of bovine inhibin (bINH1-14). Rabbits immunized subcutaneously (s.c.) or intravenously (i.v.) with EtxB::bINH1-14, with or without Freund's adjuvant, developed significant titres of antibodies that recognized an inhibin peptide fragment containing bINH1-14, native inhibins, and EtxB during separate enzyme-linked immunosorbent assay (ELISA). Passive immunization of mice with the rabbit anti-EtxB::bINH1-14 serum increased concentrations of follicle-stimulating hormone (FSH) in serum twofold compared with controls, whereas serum concentrations of luteinizing hormone (LH) were unaltered. Since FSH is the primary hormone from the pituitary gland that stimulates ovarian follicle growth and spermatogenesis, the results of this study demonstrate that EtxB::bINH1-14 has potential as antigen for development of inhibin-based fertility vaccines.     

Role of GM1 binding in the mucosal immunogenicity and adjuvant activity of the Escherichia coli heat-labile enterotoxin and its B subunit

Escherichia coli (E. coli) heat-labile toxin (LT) is a potent mucosal immunogen and immunoadjuvant towards co-administered antigens. LT is composed of one copy of the A subunit, which has ADP-ribosylation activity, and a homopentamer of B subunits, which has affinity for the toxin receptor, the ganglioside GM1. Both the ADP-ribosylation activity of LTA and GM1 binding of LTB have been proposed to be involved in immune stimulation. We investigated the roles of these activities in the immunogenicity of recombinant LT or LTB upon intranasal immunization of mice using LT/LTB mutants, lacking either ADP-ribosylation activity, GM1-binding affinity, or both. Likewise, the adjuvant properties of these LT/LTB variants towards influenza virus subunit antigen were investigated. With respect to the immunogenicity of LT and LTB, we found that GM1-binding activity is essential for effective induction of anti-LTB antibodies. On the other hand, an LT mutant lacking ADP-ribosylation activity retained the immunogenic properties of the native toxin, indicating that ADP ribosylation is not critically involved. Whereas adjuvanticity of LTB was found to be directly related to GM1-binding activity, adjuvanticity of LT was found to be independent of GM1-binding affinity. Moreover, a mutant lacking both GM1-binding and ADP-ribosylation activity, also retained adjuvanticity. These results demonstrate that neither ADP-ribosylation activity nor GM1 binding are essential for adjuvanticity of LT, and suggest an ADP-ribosylation-independent adjuvant effect of the A subunit.     

Effects of site-directed mutagenesis of Escherichia coli heat-labile enterotoxin on ADP-ribosyltransferase activity and interaction with ADP-ribosylation factors

Escherichia coli heat-labile enterotoxin (LT), an oligomeric protein with one A subunit (LTA) and five B subunits, exerts its effects via the ADP-ribosylation of Gsalpha, a guanine nucleotide-binding (G) protein that activates adenylyl cyclase. LTA also ADP-ribosylates simple guanidino compounds (e.g., arginine) and catalyzes its own auto-ADP-ribosylation. All LTA-catalyzed reactions are enhanced by ADP-ribosylation factors (ARFs), 20-kDa guanine nucleotide-binding proteins. Replacement of arginine-7 (R7K), valine-53 (V53D), serine-63 (S63K), valine 97 (V97K), or tyrosine-104 (Y104K) in LTA resulted in fully assembled but nontoxic proteins. S63K, V53D, and R7K are catalytic-site mutations, whereas V97K and Y104K are amino acid replacements adjacent to and outside of the catalytic site, respectively. The effects of mutagenesis were quantified by measuring ADP-ribosyltransferase activity (i.e., auto-ADP-ribosylation and ADP-ribosylagmatine synthesis) and interaction with ARF (i.e., inhibition of ARF-stimulated cholera toxin ADP-ribosyltransferase activity and effects of ARF on mutant auto-ADP-ribosylation). All mutants were inactive in the ADP-ribosyltransferase assay; however, auto-ADP-ribosylation in the presence of recombinant human ARF6 was detected, albeit much less than that of native LT (Y104K > V53D > V97K > R7K, S63K). Based on the lack of inhibition by free ADP-ribose, the observed auto-ADP-ribosylation activity was enzymatic and not due to the nonenzymatic addition of free ADP-ribose. V53D, S63K, and R7K were more effective than Y104K or V97K in blocking ARF stimulation of cholera toxin ADP-ribosyltransferase. Based on these data, it appears that ARF-binding and catalytic sites are not identical and that a region outside the NAD cleft may participate in the LTA-ARF interaction.     

Role of receptor binding in toxicity, immunogenicity, and adjuvanticity of Escherichia coli heat-labile enterotoxin

The role of receptor binding in the toxicity, immunogenicity, and adjuvanticity of the heat-labile enterotoxin of Escherichia coli (LT) was examined by comparing native LT and LT(G33D), a B-subunit receptor binding mutant, with respect to the ability to bind to galactose and to GM1, toxicity on mouse Y-1 adrenal tumor cells, the ability to stimulate adenylate cyclase in Caco-2 cells, enterotoxicity in the patent mouse model, and oral immunogenicity and adjuvanticity. In contrast to native LT, LT(G33D) was unable to bind to the galactosyl moiety of Sepharose 4B or GM1 but did retain the lectin-like ability to bind to immobilized galactose on 6% agarose beads. LT(G33D) had no enterotoxicity in the patent mouse model but exhibited residual toxicity on mouse Y-1 adrenal tumor cells and had an ability equivalent to that of native LT to stimulate adenylate cyclase in Caco-2 cells (5,000 versus 6,900 pmol per mg of protein). In addition, LT(G33D) was unable to serve as an effective oral adjuvant for induction of immunoglobulin G or A directed against a coadministered antigen. Furthermore, LT(G33D) elicited negligible serum and mucosal antibody responses against itself. These data indicate that the toxicity, immunogenicity, and oral adjuvanticity of LT are dependent upon binding of the B subunit to ganglioside GM1.     

Crystal structure of UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase from Escherichia coli

UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD) is a cytoplasmic enzyme involved in the biosynthesis of peptidoglycan which catalyzes the addition of D-glutamate to the nucleotide precursor UDP-N-acetylmuramoyl-L-alanine (UMA). The crystal structure of MurD in the presence of its substrate UMA has been solved to 1.9 A resolution. Phase information was obtained from multiple anomalous dispersion using the K-shell edge of selenium in combination with multiple isomorphous replacement. The structure comprises three domains of topology each reminiscent of nucleotide-binding folds: the N- and C-terminal domains are consistent with the dinucleotide-binding fold called the Rossmann fold, and the central domain with the mononucleotide-binding fold also observed in the GTPase family. The structure reveals the binding site of the substrate UMA, and comparison with known NTP complexes allows the identification of residues interacting with ATP. The study describes the first structure of the UDP-N-acetylmuramoyl-peptide ligase family.     

Glycosphingolipids as novel targets for T-cell suppression by the B subunit of recombinant heat-labile enterotoxin

Heat-labile enterotoxin subunit B (LTB) is a noncatalytic protein derived from Escherichia coli that binds to ganglioside GM1, a glycosphingolipid on the surface of mammalian cells. In this study, the effects of recombinant LTB (rLTB) on murine lymphocytes were examined in vitro. T and B cells readily bound fluorescein isothiocyanate-labeled rLTB. CD8+ T cells bound twice as much as CD4+ T cells and B cells. Exposure of T-cell subsets and B cells to rLTB abrogated mitogen-driven proliferation. CD8+ T cells were more susceptible to rLTB than either CD4+ T cells or B cells. There were differences in the sensitivity of lymphocytes from various strains of mice to rLTB. This was attributed to qualitative and quantitative differences in the CD4+ T cells. rLTB induced apoptosis in both T-cell subsets, but the level was significantly higher in CD8+ T cells. Apoptosis peaked at around 8 h after exposure to rLTB and incubation at 37 degrees C. Binding to ganglioside GM1 was essential for suppression, since rLTB/G33D, a mutant which does not bind GM1, failed to inhibit proliferation or induce apoptosis. Naive T cells, which were acutely sensitive to rLTB, became more resistant after activation. Conversely, activated T cells regained their sensitivity to rLTB when they reverted back to a resting state. A 1-h pulse with rLTB was sufficient to inhibit T-cell proliferation and cytotoxic-T-lymphocyte generation in primary mixed lymphocyte reaction cultures. CD8+ T cells were preferentially depleted in these cultures. rLTB also induced functional modifications in T cells as indicated by inhibition of gamma interferon secretion after polyclonal activation. Thus, rLTB may have immunomodulatory properties independent of its ability to induce apoptosis.     

Humoral and cellular immune responses in the murine respiratory tract following oral immunization with cholera toxin or Escherichia coli heat-labile enterotoxin

Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are the strongest mucosal immunogens identified to date and are also good adjuvants when given orally together in combination with unrelated antigens. We used these potent immunogens to monitor local and systemic immune responses following oral immunization of BALB/c mice, and compared their action on the following: (a) immunoglobulin production rates (IgG, IgM and IgA) in mucosal inductive (Peyer's patches-PPs), effector (intestinal lamina propria-LP, respiratory tract) and systemic (spleen) sites; (b) analysis of systemic antigen-specific antibodies (IgG subclasses, IgA and IgE); (c) time monitoring of fecal anti-CT and anti-LT antibodies, and (d) in vivo relevance of interleukin-6 (IL-6) to mucosal responses. Both mucosal immunogens elicited specific antibody responses (IgA, IgG) not only in the gastrointestinal tract (PP's and intestinal LP), but also in the respiratory tract and spleens of orally immunized mice. These mucosal responses were accompained by elevated secretion of IL-6 in all investigated tissues, indicating involvement of this cytokine in B-cell maturation processes. Furthermore, oral immunization with CT and LT induced elevated serum titers of IgG1 followed by IgG2a, IgG2b, IgG3 and IgA, while high antigen-specific IgA and IgG1 responses were found in fecal extracts. These findings illustrate the action of orally administered CT and LT, respectively, on several humoral and cellular immune responses not only at the gastrointestinal tract, the application site, but also in distant mucosal effector sites such as the respiratory tract. These data suggest the potential use of these mucosal adjuvants in oral immunization strategies to improve the local immune response in remote mucosal tissues, in accordance with the concept of a common mucosa-associated immune system.     

Crystal structure of Escherichia coli L-asparaginase, an enzyme used in cancer therapy

The crystal structure of Escherichia coli asparaginase II (EC 3.5.1.1), a drug (Elspar) used for the treatment of acute lymphoblastic leukemia, has been determined at 2.3 A resolution by using data from a single heavy atom derivative in combination with molecular replacement. The atomic model was refined to an R factor of 0.143. This enzyme, active as a homotetramer with 222 symmetry, belongs to the class of alpha/beta proteins. Each subunit has two domains with unique topological features. On the basis of present structural evidence consistent with previous biochemical studies, we propose locations for the active sites between the N- and C-terminal domains belonging to different subunits and postulate a catalytic role for Thr-89.     

Mucosal immunoadjuvant activity of recombinant Escherichia coli heat-labile enterotoxin and its B subunit: induction of systemic IgG and secretory IgA responses in mice by intranasal immunization with influenza virus surface antigen

The Escherichia coli heat-labile enterotoxin (LT) is a very potent mucosal immunogen. LT also has strong adjuvant activity towards coadministered unrelated antigens and is therefore of potential interest for development of mucosal vaccines. However, despite the great demand for such mucosal vaccines, the use of LT holotoxin as an adjuvant is essentially precluded by its toxicity. LT is composed of an A subunit, carrying the toxic ADP-ribosylation activity, and a pentamer of identical B subunits, which mediates binding to ganglioside GM1, the cellular receptor for the toxin. In this paper, we demonstrate that recombinant enzymatically inactive variants of LT, including the LTB pentamer by itself, retain the immunoadjuvant activity of LT holotoxin in a murine influenza model. Mice were immunized intranasally (i.n.) with influenza virus subunit antigen, consisting mostly of the isolated surface glycoprotein hemagglutinin (HA), supplemented with either recombinant LTB (rLTB), a nontoxic LT mutant (E112K, with a Glu112-->Lys substitution in the A subunit), or LT holotoxin, and the induction of systemic IgG and local S-IgA responses was evaluated by direct enzyme-linked immunosorbent assay (ELISA). Immunization with subunit antigen alone resulted in a poor systemic IgG response and no detectable S-IgA. However, supplementation of the antigen with E112K or rLTB resulted in a substantial stimulation of the serum IgG level and in induction of a strong S-IgA response in the nasal cavity. The adjuvant activity of E112K or rLTB under these conditions was essentially the same as that of the LT holotoxin. The present results demonstrate that nontoxic variants of LT, rLTB in particular, represent promising immunoadjuvants for potential application in an i.n. influenza virus subunit vaccine. Nontoxic LT variants may also be used in i.n. vaccine formulations directed against other mucosal pathogens. In this respect, it is of interest that LT(B)-stimulated antibody responses after i.n. immunization were also observed at distant mucosal sites, including the urogenital system. This, in principle, opens the possibility to develop i.n. vaccines against sexually transmitted infectious diseases.     

Crystal structure of Escherichia coli cystathionine gamma-synthase at 1.5 A resolution

The transsulfuration enzyme cystathionine gamma-synthase (CGS) catalyses the pyridoxal 5'-phosphate (PLP)-dependent gamma-replacement of O-succinyl-L-homoserine and L-cysteine, yielding L-cystathionine. The crystal structure of the Escherichia coli enzyme has been solved by molecular replacement with the known structure of cystathionine beta-lyase (CBL), and refined at 1.5 A resolution to a crystallographic R-factor of 20.0%. The enzyme crystallizes as an alpha4 tetramer with the subunits related by non-crystallographic 222 symmetry. The spatial fold of the subunits, with three functionally distinct domains and their quaternary arrangement, is similar to that of CBL. Previously proposed reaction mechanisms for CGS can be checked against the structural model, allowing interpretation of the catalytic and substrate-binding functions of individual active site residues. Enzyme-substrate models pinpoint specific residues responsible for the substrate specificity, in agreement with structural comparisons with CBL. Both steric and electrostatic designs of the active site seem to achieve proper substrate selection and productive orientation. Amino acid sequence and structural alignments of CGS and CBL suggest that differences in the substrate-binding characteristics are responsible for the different reaction chemistries. Because CGS catalyses the only known PLP-dependent replacement reaction at Cgamma of certain amino acids, the results will help in our understanding of the chemical versatility of PLP.     

Mutations in the A subunit affect yield, stability, and protease sensitivity of nontoxic derivatives of heat-labile enterotoxin

Heat-labile toxin (LT) is a protein related to cholera toxin, produced by enterotoxigenic Escherichia coli strains, that is organized as an AB5 complex. A number of nontoxic derivatives of LT, useful for new or improved vaccines against diarrheal diseases or as mucosal adjuvants, have been constructed by site-directed mutagenesis. Here we have studied the biochemical properties of the nontoxic mutants LT-K7 (Arg-7-->Lys), LT-D53 (Val-53-->Asp), LT-K63 (Ser-63-->Lys), LT-K97 (Val-97-->Lys), LT-K104 (Tyr-104-->Lys), LT-K114 (Ser-114-->Lys), and LT-K7/K97 (Arg-7-->Lys and Val-97-->Lys). We have found that mutations in the A subunit may have profound effects on the ability to form the AB5 structure and on the stability and trypsin sensitivity of the purified proteins. Unstable mutants, during long-term storage at 4 degrees C, showed a decrease in the amount of the assembled protein in solution and a parallel appearance of soluble monomeric B subunit. This finding suggests that the stability of the B pentamer is influenced by the A subunit which is associated with it. Among the seven nontoxic mutants tested, LT-K63 was found to be efficient in AB5 production, extremely stable during storage, resistant to proteolytic attack, and very immunogenic. In conclusion, LT-K63 is a good candidate for the development of antidiarrheal vaccines and mucosal adjuvants.     

Crystal structure of intact elongation factor EF-Tu from Escherichia coli in GDP conformation at 2.05 A resolution

The crystal structure of intact elongation factor Tu (EF-Tu) from Escherichia coli in GDP-bound conformation has been determined using a combination of multiple isomorphous replacement (MIR) and multiwavelength anomalous diffraction (MAD) methods. The current atomic model has been refined to a crystallographic R factor of 20.3 % and free R-factor of 26.8 % in the resolution range of 10-2.05 A. The protein consists of three domains: domain 1 has an alpha/beta structure; while domain 2 and domain 3 are beta-barrel structures. Although the global fold of the current model is similar to those of published structures, the secondary structural assignment has been improved due to the high quality of the current model. The switch I region (residues 40-62) is well ordered in this structure. Comparison with the structure of EF-Tu in GDP-bound form from Thermus aquaticus shows that although the individual domain structures are similar in these two structures, the orientation of domains changes significantly. Interactions between domains 1 and 3 in our E. coli EF-Tu-GDP complex are quite different from those of EF-Tu with bound GTP from T. aquaticus, due to the domain rearrangement upon GTP binding. The binding sites of the Mg2+ and guanine nucleotide are revealed in detail. Two water molecules that co-ordinate the Mg2+ have been identified to be well conserved in the GDP and GTP-bound forms of EF-Tu structures, as well as in the structure of Ras p21 with bound GDP. Comparisons of the Mg2+ binding site with other guanine nucleotide binding proteins in GDP-bound forms show that the Mg2+ co-ordination patterns are well preserved among these structures.     

Crystal structure of the ATP-binding subunit of an ABC transporter

ABC transporters (also known as traffic ATPases) form a large family of proteins responsible for the translocation of a variety of compounds across membranes of both prokaryotes and eukaryotes. The recently completed Escherichia coli genome sequence revealed that the largest family of paralogous E. coli proteins is composed of ABC transporters. Many eukaryotic proteins of medical significance belong to this family, such as the cystic fibrosis transmembrane conductance regulator (CFTR), the P-glycoprotein (or multidrug-resistance protein) and the heterodimeric transporter associated with antigen processing (Tap1-Tap2). Here we report the crystal structure at 1.5 A resolution of HisP, the ATP-binding subunit of the histidine permease, which is an ABC transporter from Salmonella typhimurium. We correlate the details of this structure with the biochemical, genetic and biophysical properties of the wild-type and several mutant HisP proteins. The structure provides a basis for understanding properties of ABC transporters and of defective CFTR proteins.     

Interaction of ADP-ribosylation factor with Escherichia coli enterotoxin that contains an inactivating lysine 112 substitution

Cholera toxin and Escherichia coli heat-labile enterotoxin (LT) exert their effects on cells through ADP-ribosylation of guanine nucleotide-binding proteins. Both toxins consist of one A subunit, which is an ADP-ribosyltransferase, and five B (or binding) subunits. Their enzymatic activities are latent; activation requires reduction and proteolysis, resulting in a catalytically active A1 protein and a much smaller A2 protein. These ADP-ribosyltransferases are activated by GTP-dependent 20-kDa ADP-ribosylation factors or ARFs. To determine if proteolysis plus reduction is required for appearance of the ARF allosteric site as well as for catalytic activity, an inactive mutant of LT, LT(E112K), with replacement of glutamate by lysine at position 112 of its A subunit, was utilized as a competitor in cholera toxin ADP-ribosyltransferase assays containing limiting amounts of ARF. LT(E112K) required trypsinization and reduction to become a potent, concentration-dependent inhibitor. Inhibition was reversed by increasing concentrations of ARF. Reduction or trypsinization alone did not generate an inhibitory form of LT(E112K). These studies are consistent with the conclusion that the ARF site is not expressed in the latent toxin. Both trypsinization and reduction are required for expression of a functional ARF binding site as well as for catalytic activity.     

The crystal structure of the sevenfold mutant of barley beta-amylase with increased thermostability at 2.5 A resolution

The three-dimensional structure of the sevenfold mutant of barley beta-amylase (BBA-7s) with increased thermostability was determined by X-ray crystallography. The enzyme was purified as a single component and crystallized by a hanging drop method in the presence of 14 % PEG 6000. The crystals belong to space group P43212 with cell dimensions a=b=72.11 A, c=250.51 A. The diffraction data up to 2.5 A were collected after soaking the crystal in 100 mM maltose with Rsym of 8.6 %. The structure was determined by a molecular replacement method using soybean beta-amylase (SBA) as a search model and refined to an R-factor of 18.7 %. The final model included 500 amino acid residues, 141 water molecules and three glucose residues, which were located at subsites 1-2 and 4 in the active site. The r.m.s. distance of 485 Calpha atoms between BBA-7s and SBA was 0.62 A. Out of the seven mutated amino acids, four (Ser295Ala, Ile297Val, Ser351Pro and Ala376Ser) were substitutions from the common residues with SBA to the thermostable forms. A comparison of the structures of BBA-7s and SBA indicated that the side-chain of Ser376 makes new hydrogen bonds to the main-chain of an adjacent beta-strand, and that the side-chains of Val297 reduce an unfavorable interaction between the side-chains of Ala314. The mutation of Ser295Ala breaks the hydrogen bond between Ser295 OG and Tyr195 OH, which seems to be the reason for the unoccupied glucose residue at subsite 3. The tandem mutations at 350-352 including substitutions to two Pro residues suggested the reduction of main-chain entropy in the unfolded structure of this solvent-exposed protruded loop.