Structural investigation of the biosynthesis of alternative lower ligands for cobamides by nicotinate mononucleotide: 5,6-dimethylbenzimidazole phosphoribosyltransferase from Salmonella enterica.

Nicotinate mononucleotide (NaMN):5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella enterica plays a central role in the synthesis of alpha-ribazole, a key component of the lower ligand of cobalamin. Surprisingly, CobT can phosphoribosylate a wide range of aromatic substrates, giving rise to a wide variety of lower ligands in cobamides. To understand the molecular basis for this lack of substrate specificity, the x-ray structures of CobT complexed with adenine, 5-methylbenzimidazole, 5-methoxybenzimidazole, p-cresol, and phenol were determined. Furthermore, adenine, 5-methylbenzimidazole, 5-methoxybenzimidazole, and 2-hydroxypurine were observed to react with NaMN within the crystal lattice and undergo the phosphoribosyl transfer reaction to form product. Significantly, the stereochemistries of all products are identical to those found in vivo. Interestingly, p-cresol and phenol, which are the lower ligand in Sporomusa ovata, bound to CobT but did not react with NaMN. This study provides a structural explanation for how CobT can phosphoribosylate most of the commonly observed lower ligands found in cobamides with the exception of the phenolic lower ligands observed in S. ovata. This is accomplished with minor conformational changes in the side chains that constitute the 5,6-dimethylbenzimidazole binding site. These investigations are consistent with the implication that the nature of the lower ligand is controlled by metabolic factors rather by the specificity of the phosphoribosyltransferase.     

Structure of acetylcholinesterase complexed with (-)-galanthamine at 2.3 A resolution

In recent years, the actin cytoskeleton in Schizosaccharomyces pombe has become the subject of intense scrutiny. However, to date, only a single actin mutation has been identified. Described here is the isolation and characterization of four new cold-sensitive actin mutations. Sequence analysis of the mutant actin genes indicated that each of these mutations caused alterations in single amino acids that are conserved in all actin sequences. These mutants differ in their phenotypes. One of these mutations (act1-48) was identified as an extragenic suppressor of a mutation in the cdc4 gene, which is required for actin ring formation and cytokinesis. Interestingly, when act1-48 mutant cells were shifted to the restrictive temperature, actin patches were not detected but the actin ring formation and stability was unaffected. The three other mutations, act1-16, act1-32 and act1-67, primarily affected the actin ring formation or stability while F-actin patches did not seem to be substantially different in appearance. Given that the ultrastructural architectures of F-actin patches and the F-actin ring are presently unclear, these mutations, which affect one structure or the other, should be useful for future studies on the role of actin itself in the function of these F-actin-containing structures in S. pombe.     

The cobT gene of Salmonella typhimurium encodes the NaMN: 5,6-dimethylbenzimidazole phosphoribosyltransferase responsible for the synthesis of N1-(5-phospho-alpha-D-ribosyl)-5,6-dimethylbenzimidazole, an intermediate in the synthesis of the nucleotide loop of cobalamin.

We present in vitro evidence which demonstrates that CobT is the nicotinate nucleotide:5,6-dimethylbenzimidazole (DMB) phosphoribosyltransferase (EC 2.4.2.21) that catalyzes the synthesis of N1-(5-phospho-alpha-D-ribosyl)-5,6-dimethylbenzimidazole, a biosynthetic intermediate of the pathway that assembles the nucleotide loop of cobalamin in Salmonella typhimurium. Mutants previously isolated as DMB auxotrophs are shown by physical and genetic mapping studies and complementation studies to carry lesions in cobT. Explanations for this unexpected phenotype of cobT mutants are discussed. The expected nucleotide loop assembly phenotype of cobT mutants can be observed only in a specific genetic background, i.e., cobB deficient, an observation that is consistent with the existence of an alternative CobT function (G. A. O'Toole, M. R. Rondon, and J. C. Escalante-Semerena, J. Bacteriol. 175:3317-3326, 1993). Computer analysis of CobT homologs showed that at the amino acid level, enteric CobT proteins were 80% identical whereas Pseudomonas denitrificans and Rhizobium meliloti CobT proteins were 95% identical. Interestingly, the degree of identity between enteric and nonenteric CobT homologs was only 30%. The same pattern of homologies was reported for the S. typhimurium CobA, Escherichia coli BtuR, and P. denitrificans CobO proteins (S.-J. Suh and J.C. Escalante-Semerena, Gene 129:93-97, 1993), suggesting evolutionary divergence between the cob genes found in the enteric bacteria E. coli and S. typhimurium and those found in P. denitrificans and R. meliloti.     

Crystallization and preliminary X-ray diffraction analysis of an acidic phospholipase A(2) complexed with p-bromophenacyl bromide and alpha-tocopherol inhibitors at 1.9- and 1.45-A resolution

An acidic phospholipase A(2) (PLA(2)) isolated from Bothrops jararacussu snake venom was crystallized with two inhibitors: alpha-tocopherol (vitamin E) and p-bromophenacyl bromide (BPB). The crystals diffracted at 1.45- and 1.85-A resolution, respectively, for the complexes with alpha-tocopherol and p-bromophenacyl bromide. The crystals are not isomorphous with those of the native protein, suggesting the inhibitors binding was successful and changes in the quaternary structure may have occurred.     

A series of crystal structures of a meta-cleavage product hydrolase from Pseudomonas fluorescens IP01 (CumD) complexed with various cleavage products

Meta-cleavage product hydrolase (MCP-hydrolase) is one of the key enzymes in the microbial degradation of aromatic compounds. MCP-hydrolase produces 2-hydroxypenta-2,4-dienoate and various organic acids, according to the C6 substituent of the substrate. Comprehensive analysis of the substrate specificity of the MCP-hydrolase from Pseudomonas fluorescens IP01 (CumD) was carried out by determining the kinetic parameters for nine substrates and crystal structures complexed with eight cleavage products. CumD preferred substrates with long non-branched C6 substituents, but did not effectively hydrolyze a substrate with a phenyl group. Superimposition of the complex structures indicated that benzoate was bound in a significantly different direction than other aliphatic cleavage products. The directions of the bound organic acids appeared to be related with the k(cat) values of the corresponding substrates. The Ile139 and Trp143 residues on helix alpha4 appeared to cause steric hindrance with the aromatic ring of the substrate, which hampers base-catalyzed attack by water.     

The hair-dye reagent 2-(2',4'-diaminophenoxy)ethanol is mutagenic to Salmonella typhimurium

A new hair-dye ingredient, 2-(2',4'-diaminophenoxy)ethanol (2,4-DAPE), was described as being devoid of any genotoxic activity on the basis of a multi-laboratory study. Since 2,4-DAPE is a close analogue of 2,4-diaminoanisole (2,4-DAA), which is mutagenic and carcinogenic, we tested this claim by assaying 2,4-DAPE for bacterial mutagenicity. Two samples of 2,4-DAPE X 2HCl were synthesized by reduction of the corresponding dinitrophenoxyethanol and identity and purity were established by elemental analysis, NMR spectrometry, mass-spectrometry, UV-spectrophotometry, TLC and HPLC. Fresh aqueous solutions of 2,4-DAPE X 2HCl were assayed in several separate plate tests using S. typhimurium TA1538, TA97, TA98 and TA100, and E. coli WP2uvrA (pKM101), 3 plates per dose and 0%, 4%, 10% and 30% Aroclor 1254-induced rat-liver S9 in S9 mixes. We obtained negative results in TA100 and E. coli. Reproducible, statistically significant dose-related increases in revertants (up to 14 times the background) were obtained in frame-shift mutants of S. typhimurium in the dose range 10-80 micrograms per plate. Mutagenicity was S9-dependent, significant increases in revertants being obtained only with 50 microliter per plate or more of S9. 2,4-DAPE induced significant mutagenic effects at doses of less than 1 micrograms per ml in TA1538 and TA98 in fluctuation tests using 2% S9 in the S9 mix. In plate tests, 2,4-DAPE was less mutagenic (by a factor of about 8) than 2,4-DAA, which gave the highest mutant yields with 20 microliter S9 per plate (4% S9 in the S9 mix). 2,4-DAPE obtained commercially was about 8 times more mutagenic than our sample of 2,4-DAPE. After purification, the commercial product, now chromatographically identical with our own sample, gave plate-test results close to those obtained for our samples of 2,4-DAPE. A review of the published reports (in which 2,4-DAPE was claimed to be inactive in a variety of short-term tests) revealed: (a) the use of protocols for bacterial mutagenicity testing which, in the light of our own results, were probably too limited in scope, especially in the choice of conditions for metabolic activation; (b) insufficient information on the identification and purity of the samples of 2,4-DAPE tested in the published collaborative study.     

High resolution crystal structures of triosephosphate isomerase complexed with its suicide inhibitors: The conformational flexibility of the catalytic glutamate in its closed, liganded active site

The key residue of the active site of triosephosphate isomerase (TIM) is the catalytic glutamate, which is proposed to be important (i) as a catalytic base, for initiating the reaction, as well as (ii) for the subsequent proton shuttling steps. The structural properties of this glutamate in the liganded complex have been investigated by studying the high resolution crystal structures of typanosomal TIM, complexed with three suicide inhibitors: (S)-glycidol phosphate ((S)-GOP, at 0.99 Å resolution), (R)-glycidol phosphate, ((R)-GOP, at 1.08 Å resolution), and bromohydroxyacetone phosphate (BHAP, at 1.97 Å resolution). The structures show that in the (S)-GOP active site this catalytic glutamate is in the well characterized, competent conformation. However, an unusual side chain conformation is observed in the (R)-GOP and BHAP complexes. In addition, Glu97, salt bridged to the catalytic lysine in the competent active site, adopts an unusual side chain conformation in these two latter complexes. The higher chemical reactivity of (S)-GOP compared with (R)-GOP, as known from solution studies, can be understood: the structures indicate that in the case of (S)-GOP, Glu167 can attack the terminal carbon of the epoxide in a stereoelectronically favored, nearly linear O–C–O arrangement, but this is not possible for the (R)-GOP isomer. These structures confirm the previously proposed conformational flexibility of the catalytic glutamate in its closed, liganded state. The importance of this conformational flexibility for the proton shuttling steps in the TIM catalytic cycle, which is apparently achieved by a sliding motion of the side chain carboxylate group above the enediolate plane, is also discussed.     

X-ray structures of Torpedo californica acetylcholinesterase complexed with (+)-huperzine A and (-)-huperzine B: structural evidence for an active site rearrangement

Kinetic and structural data are presented on the interaction with Torpedo californica acetylcholinesterase (TcAChE) of (+)-huperzine A, a synthetic enantiomer of the anti-Alzheimer drug, (-)-huperzine A, and of its natural homologue (-)-huperzine B. (+)-Huperzine A and (-)-huperzine B bind to the enzyme with dissociation constants of 4.30 and 0.33 microM, respectively, compared to 0.18 microM for (-)-huperzine A. The X-ray structures of the complexes of (+)-huperzine A and (-)-huperzine B with TcAChE were determined to 2.1 and 2.35 A resolution, respectively, and compared to the previously determined structure of the (-)-huperzine A complex. All three interact with the "anionic" subsite of the active site, primarily through pi-pi stacking and through van der Waals or C-H.pi interactions with Trp84 and Phe330. Since their alpha-pyridone moieties are responsible for their key interactions with the active site via hydrogen bonding, and possibly via C-H.pi interactions, all three maintain similar positions and orientations with respect to it. The carbonyl oxygens of all three appear to repel the carbonyl oxygen of Gly117, thus causing the peptide bond between Gly117 and Gly118 to undergo a peptide flip. As a consequence, the position of the main chain nitrogen of Gly118 in the "oxyanion" hole in the native enzyme becomes occupied by the carbonyl of Gly117. Furthermore, the flipped conformation is stabilized by hydrogen bonding of Gly117O to Gly119N and Ala201N, the other two functional elements of the three-pronged "oxyanion hole" characteristic of cholinesterases. All three inhibitors thus would be expected to abolish hydrolysis of all ester substrates, whether charged or neutral.     

The reaction of indole with the aminoacrylate intermediate of Salmonella typhimurium tryptophan synthase: observation of a primary kinetic isotope effect with 3-[(2)H]indole

The bacterial tryptophan synthase alpha(2)beta(2) complex catalyzes the final reactions in the biosynthesis of L-tryptophan. Indole is produced at the active site of the alpha-subunit and is transferred through a 25-30 A tunnel to the beta-active site, where it reacts with an aminoacrylate intermediate. Lane and Kirschner proposed a two-step nucleophilic addition-tautomerization mechanism for the reaction of indole with the aminoacrylate intermediate, based on the absence of an observed kinetic isotope effect (KIE) when 3-[(2)H]indole reacts with the aminoacrylate intermediate. We have now observed a KIE of 1.4-2.0 in the reaction of 3-[(2)H]indole with the aminoacrylate intermediate in the presence of monovalent cations, but not when an alpha-subunit ligand, disodium alpha-glycerophosphate (Na(2)GP), is present. Rapid-scanning stopped flow kinetic studies were performed of the reaction of indole and 3-[(2)H]indole with tryptophan synthase preincubated with L-serine, following the decay of the aminoacrylate intermediate at 350 nm, the formation of the quinonoid intermediate at 476 nm, and the formation of the L-Trp external aldimine at 423 nm. The addition of Na(2)GP dramatically slows the rate of reaction of indole with the alpha-aminoacrylate intermediate. A primary KIE is not observed in the reaction of 3-[(2)H]indole with the aminoacrylate complex of tryptophan synthase in the presence of Na(2)GP, suggesting binding of indole with tryptophan synthase is rate limiting under these conditions. The reaction of 2-methylindole does not show a KIE, either in the presence of Na(+) or Na(2)GP. These results support the previously proposed mechanism for the beta-reaction of tryptophan synthase, but suggest that the rate limiting step in quinonoid intermediate formation from indole and the aminoacrylate intermediate is deprotonation.     

The course of phosphorus in the reaction of N-acetyl-L-glutamate kinase,determined from the structures of crystalline complexes,including a complex with an AlF(4)(-) transition state mimic

N-Acetyl-L-glutamate kinase (NAGK), the structural paradigm of the enzymes of the amino acid kinase family, catalyzes the phosphorylation of the gamma-COO(-) group of N-acetyl-L-glutamate (NAG) by ATP. We determine here the crystal structures of NAGK complexes with MgADP, NAG and the transition-state analog AlF(4)(-); with MgADP and NAG; and with ADP and SO(4)(2-). Comparison of these structures with that of the MgAMPPNP-NAG complex allows to delineate three successive steps during phosphoryl transfer: at the beginning, when the attacking and leaving O atoms and the P atom are imperfectly aligned and the distance between the attacking O atom and the P atom is 2.8A; midway, at the bipyramidal intermediate, with nearly perfect alignment and a distance of 2.3A; and, when the transfer is completed. The transfer occurs in line and is strongly associative, with Lys8 and Lys217 stabilizing the transition state and the leaving group, respectively, and with Lys61, in contrast with an earlier proposal, not being involved. Three water molecules found in all the complexes play, together with Asp162 and the Mg, crucial structural roles. Two glycine-rich loops (beta1-alphaA and beta2-alphaB) are also very important, moving in the different complexes in concert with the ligands, to which they are hydrogen-bonded, either locking them in place for reaction or stabilizing the transition state. The active site is too narrow to accommodate the substrates without compressing the reacting groups, and this compressive strain appears a crucial component of the catalytic mechanism of NAGK, and possibly of other enzymes of the amino acid kinase family such as carbamate kinase. Initial binding of the two substrates would require a different enzyme conformation with a wider active site, and the energy of substrate binding would be used to change the conformation of the active center, causing substrate strain towards the transition state.     

The transition of bovine trypsinogen to a trypsin-like state upon strong ligand binding. The refined crystal structures of the bovine trypsinogen-pancreatic trypsin inhibitor complex and of its ternary complex with Ile-Val at 1.9 A resolution

The complex formed by bovine trypsinogen and the pancreatic trypsin inhibitor crystallizes in large crystals isomorphous with trypsin-PTI² complex crystals Rühlmann et al. 1973. X-ray diffraction data to 1.9 Å resolution were collected in the absence and presence of Ile-Val dipeptide. Both trypsinogen complex structures have been crystallographically refined, using the refined trypsin-PTI complex Huber et al. 1974a as a starting model. The final R values are 0.25 and 0.26, respectively. The mean main-chain atom deviations between the three complex structures are about 0.15 Å. In contrast, the mean deviation between the complexed and the free trypsinogen Fehlhammer et al. 1977 is 0.28 Å, reflecting the influence of crystal packing and complexation. The trypsinogen component adopts a trypsin-like conformation upon PTI binding: The Asp194 side-chain turns around and the activation domain becomes rigid, forming the specificity pocket and the Ile16 binding cleft. The specific interactions between PTI and trypsin are also observed in the trypsinogen complex. As in free trypsinogen, the N-terminus including residues Val10 to Gly18 is mobile and sticks out into solution. Apart from the different arrangement of the N-termini in the two complexes, the only significant, but minor structural difference is the enhanced thermal mobility of the autolysis loop in the trypsinogen complex. Upon binding of the Ile-Val dipeptide, the autolysis loop becomes fixed as in the trypsin complex. The Ile-Val position is identical in the ternary and the trypsin complex.     

A Salmonella typhimurium strain genetically engineered to secrete effectively a bioactive human interleukin (hIL)-6 via the Escherichia coli hemolysin secretion apparatus

Human interleukin-6 (hIL-6) cDNA was genetically fused with the Escherichia coli hemolysin secretorial signal ( hlyA_S ) sequence in a plasmid vector. Recombinant E. coli XL-1 Blue and attenuated Salmonella typhimurium secreted a 30 kDa hIL-6-HlyA_S fusion protein, with an additional form of higher apparent molecular mass produced by S. typhimurium . In S. typhimurium cultures hIL-6-HlyA_S concentrations entered a plateau at 500 to 600 ng ml⁻¹ culture supernatant. In contrast to E. coli XL-1 Blue, in S. typhimurium culture supernatants hIL-6-HlyA_S was accumulated faster reaching three-fold higher maximal concentrations. The cell proliferating activity of hIL-6-HlyA_S fusion protein(s) was equivalent to that of mature recombinant hIL-6. Furthermore, hIL-6-secreting S. typhimurium were less invasive than the attenuated control strain. Therefore, the bulky hemolysin secretorial peptide at the C-terminus of the fusion protein does not markably affect hIL-6 activity, suggesting that the hemolysin secretion apparatus provides an excellent system to study immunomodulatory effects of in situ synthesized IL-6 in Salmonella vaccine strains.     

The three-dimensional structure of the bifunctional proteinase K/alpha-amylase inhibitor from wheat (PK13) at 2.5 A resolution

Wheat germ contains an inhibitor for proteinase K, called PK13 (Mr approximately 19,600) which simultaneously inhibits alpha-amylase. PK13 was crystallized, space group P21, a = 43.02 (5) A, b = 65.18 (7) A, c = 32.33 (4) A, beta = 112.79 degrees (9), X-ray data were collected to 2.5 A resolution, the structure solved by molecular replacement on the basis of the atomic coordinates of the homologous Erythrina caffra DE-3 inhibitor, and refined with simulated annealing techniques with a current R-factor of 21%. The three-dimensional structure of PK13 is stabilised by two disulfide bridges and has a central beta-barrel with distorted beta-structure. In analogy to related inhibitors, the binding site for proteinase K is assumed to be located on the surface of the protein (amino acid residues 66-67), although the 75-76 peptide bond is cleaved upon binding.     

Structure of mammalian cytochrome P450 2B4 complexed with 4-(4-chlorophenyl)imidazole at 1.9-A resolution: insight into the range of P450 conformations and the coordination of redox partner binding

A 1.9-A molecular structure of the microsomal cytochrome P450 2B4 with the specific inhibitor 4-(4-chlorophenyl)imidazole (CPI) in the active site was determined by x-ray crystallography. In contrast to the previous experimentally determined 2B4 structure, this complex adopted a closed conformation similar to that observed for the mammalian 2C enzymes. The differences between the open and closed structures of 2B4 were primarily limited to the lid domain of helices F through G, helices B' and C, the N terminus of helix I, and the beta(4) region. These large-scale conformational changes were generally due to the relocation of conserved structural elements toward each other with remarkably little remodeling at the secondary structure level. For example, the F' and G' helices were maintained with a sharp turn between them but are placed to form the exterior ceiling of the active site in the CPI complex. CPI was closely surrounded by residues from substrate recognition sites 1, 4, 5, and 6 to form a small, isolated hydrophobic cavity. The switch from open to closed conformation dramatically relocated helix C to a more proximal position. As a result, heme binding interactions were altered, and the putative NADPH-cytochrome P450 reductase binding site was reformed. This suggests a structural mechanism whereby ligand-induced conformational changes may coordinate catalytic activity. Comparison of the 2B4/CPI complex with the open 2B4 structure yields insights into the dynamics involved in substrate access, tight inhibitor binding, and coordination of substrate and redox partner binding.     

Crystal structure of macrophage migration inhibitory factor complexed with (E)-2-fluoro-p-hydroxycinnamate at 1.8 A resolution: implications for enzymatic catalysis and inhibition.

Macrophage migration inhibitory factor (MIF) exhibits dual activities. It acts as an immunoregulatory protein as well as a phenylpyruvate tautomerase. To understand better the relationship between these two activities and to elucidate the structural basis for the enzymatic activity, a crystal structure of a complex between murine MIF and (E)-2-fluoro-p-hydroxycinnamate, a competitive inhibitor of the tautomerase activity, has been determined to 1.8 A resolution. The structure is nearly superimposable on that of the free protein indicating that the presence of the inhibitor does not result in any major structural changes. The inhibitor also confirms the location of the active site in a hydrophobic cavity containing the amino-terminal proline. Within this cavity, the inhibitor interacts with residues from adjacent subunits. At the back of the cavity, the side-chain carbonyl oxygen of Asn-97' interacts with the phenolic hydroxyl group of the inhibitor while at the mouth of the cavity the ammonium group of Lys-32 interacts with a carboxylate oxygen. The other carboxylate oxygen of the inhibitor interacts with Pro-1. The hydroxyl group of Tyr-95' interacts weakly with the fluoro group on the inhibitor. The hydrophobic side chains of five active-site residues (Met-2, Ile-64, Met-101, Val-106, and Phe-113) and the phenyl moiety of Tyr-95' are responsible for the binding of the phenyl group. Further insight into the enzymatic activity of MIF was obtained by carrying out kinetic studies using the enol isomers of phenylpyruvate and (p-hydroxyphenyl)pyruvate. The results demonstrate that MIF processes the enol isomers more efficiently than the keto isomers primarily because of a decrease in Km. On the basis of these results, a mechanism is proposed for the MIF-catalyzed tautomerization reaction.     

The Salmonella pathogenicity island (SPI)-2 and SPI-1 type III secretion systems allow Salmonella serovar typhimurium to trigger colitis via MyD88-dependent and MyD88-independent mechanisms

Salmonella typhimurium can colonize the gut, invade intestinal tissues, and cause enterocolitis. In vitro studies suggest different mechanisms leading to mucosal inflammation, including 1) direct modulation of proinflammatory signaling by bacterial type III effector proteins and 2) disruption or penetration of the intestinal epithelium so that penetrating bacteria or bacterial products can trigger innate immunity (i.e., TLR signaling). We studied these mechanisms in vivo using streptomycin-pretreated wild-type and knockout mice including MyD88(-/-) animals lacking an adaptor molecule required for signaling via most TLRs. The Salmonella SPI-1 and the SPI-2 type III secretion systems (TTSS) contributed to inflammation. Mutants that retain only a functional SPI-1 (M556; sseD::aphT) or a SPI-2 TTSS (SB161; DeltainvG) caused attenuated colitis, which reflected distinct aspects of the colitis caused by wild-type S. typhimurium: M556 caused diffuse cecal inflammation that did not require MyD88 signaling. In contrast, SB161 induced focal mucosal inflammation requiring MyD88. M556 but not SB161 was found in intestinal epithelial cells. In the lamina propria, M556 and SB161 appeared to reside in different leukocyte cell populations as indicated by differential CD11c staining. Only the SPI-2-dependent inflammatory pathway required aroA-dependent intracellular growth. Thus, S. typhimurium can use two independent mechanisms to elicit colitis in vivo: SPI-1-dependent and MyD88-independent signaling to epithelial cells and SPI-2-dependent intracellular proliferation in the lamina propria triggering MyD88-dependent innate immune responses.     

ET-1-induced pulmonary vasoconstriction shifts from ET(A)- to ET(B)-receptor-mediated reaction after preconstriction.

Endothelin-1 (ET-1) has been reported to induce pulmonary vasoconstriction via either ET(A) or ET(B) receptors, and vasorelaxation after ET-1 injection has been observed. Our study investigated the effects of ET-1 in isolated rabbit lungs, which were studied at basal tone (part I) and after preconstriction (U-46619; part II). Pulmonary arterial pressure (PAP) and lung weight gain were monitored continuously. In part I, ET-1 (10(-8) M; n = 6; control) was injected after pretreatment with the ET(A)-receptor antagonist BQ-123 (10(-6) M; n = 6) or the ET(B)-receptor antagonist BQ-788 (10(-6) M; n = 6). The same protocol was carried out in part II after elevation of pulmonary vascular tone. ET-1 induced an immediate PAP increase (DeltaPAP 4.3 +/- 0.4 mmHg at 10 min) that was attenuated by pretreatment with BQ-123 (P < 0.05 at 10 min and P < 0.01 thereafter) and that was more pronounced after BQ-788 (P < 0.01 at 10 min and P < 0.001 thereafter). In part II, ET-1 induced an immediate rise in PAP with a maximum after 5 min (DeltaPAP 6.3 +/- 1.4 mmHg), leveling off at DeltaPAP 3.2 +/- 0.2 mmHg after 15 min. Pretreatment with BQ-123 failed to attenuate the increase. BQ-788 significantly reduced the peak pressure at 5 min (0.75 +/- 0.4 mmHg; P < 0.001) as well as the plateau pressure thereafter (P < 0.01). We conclude that ET-1 administration causes pulmonary vasoconstriction independent of basal vascular tone, and, at normal vascular tone, the vasoconstriction seems to be mediated via ET(A) receptors. BQ-788 treatment resulted in even more pronounced vasoconstriction. After pulmonary preconstriction, ET(A) antagonism exerted no effects on PAP, whereas ET(B) antagonism blocked the PAP increase. Therefore, ET-1-induced pulmonary vasoconstriction is shifted from an ET(A)-related to an ET(B)-mediated mechanism after pulmonary vascular preconstriction.     

Structural basis for enantiomer binding and separation of a common beta-blocker: crystal structure of cellobiohydrolase Cel7A with bound (S)-propranolol at 1.9 A resolution

Cellobiohydrolase Cel7A (previously called CBH 1), the major cellulase produced by the mould fungus Trichoderma reesei, has been successfully exploited as a chiral selector for separation of stereo-isomers of some important pharmaceutical compounds, e.g. adrenergic beta-blockers. Previous investigations, including experiments with catalytically deficient mutants of Cel7A, point unanimously to the active site as being responsible for discrimination of enantiomers.In this work the structural basis for enantioselectivity of basic drugs by Cel7A has been studied by X-ray crystallography. The catalytic domain of Cel7A was co-crystallised with the (S)-enantiomer of a common beta-blocker, propranolol, at pH 7, and the structure of the complex was determined and refined at 1. 9 A resolution. Indeed, (S)-propranolol binds at the active site, in glucosyl-binding subsites -1/+1. The catalytic residues Glu212 and Glu217 make tight salt links with the secondary amino group of (S)-propranolol. The oxygen atom attached to the chiral centre of (S)-propranolol forms hydrogen bonds to the nucleophile Glu212 O(epsilon1) and to Gln175 N(epsilon2), whereas the aromatic naphthyl moiety stacks with the indole ring of Trp376 in site +1. The bidentate charge interaction with the catalytic glutamate residues is apparently crucial, since no enantioselectivity has been obtained with the catalytically deficient mutants E212Q and E217Q.Activity inhibition experiments with wild-type Cel7A were performed in conditions close to those used for crystallisation. Competitive inhibition constants for (R)- and (S)-propranolol were determined at 220 microM and 44 microM, respectively, corresponding to binding free energies of 20 kJ/mol and 24 kJ/mol, respectively. The K(i) value for (R)-propranolol was 57-fold lower than the highest concentration, 12.5 mM, used in co-crystallisation experiments. Still several attempts to obtain a complex with the (R)-enantiomer have failed.By using cellobiose as a selective competing ligand, the retention of the enantiomers of propranolol on the chiral stationary phase (CSP) based on Cel7A mutant D214N were resolved into enantioselective and non- selective binding. The enantioselective binding was weaker for both enantiomers on D214N-CSP than on wild-type-CSP.     

The crystal structure of DR2241 from Deinococcus radiodurans at 1.9 A resolution reveals a multi-domain protein with structural similarity to chelatases but also with two additional novel domains

A unique family of proteins have been identified in the Deinococcus genus with an N-terminal cobalamin (vitamin B(12)) chelatase domain denoted CbiX and an additional unique C-terminal domain with unknown function. Here we report the first crystal structure from this new family of proteins with the structure of Deinococcus radiodurans protein DR2241. The structure reveals a multi-domain protein where domains A (residues 1-132) has the same fold as the small CbiX (CbiX(S)), domains A and B (residues 1-272) follow the chelatase super-family fold and the two additional unique domains C and D have no structural homologues. Domain D harbours the sequence motifs CxxC and CxxxC, in which DR2241 gives the first evidence that these motifs bind a [4Fe-4S] iron-sulphur cluster. In solution there are indications of multimeric forms, and in the crystallographic asymmetric unit a tetramer is found where domains C and D are involved in stabilising the tetrameric assembly.