Crystal structures of KDOP synthase in its binary complexes with the substrate phosphoenolpyruvate and with a mechanism-based inhibitor.

The crystal structures of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDOPS) from Escherichia coli complexed with the substrate phosphoenolpyruvate (PEP) and with a mechanism-based inhibitor (K(d) = 0.4 microM) were determined by molecular replacement using X-ray diffraction data to 2.8 and 2.3 A resolution, respectively. Both the KDOPS.PEP and KDOPS.inhibitor complexes crystallize in the cubic space group I23 with cell constants a = b = c = 117.9 and 117.6 A, respectively, and one subunit per asymmetric unit. The two structures are nearly identical, and superposition of their Calpha atoms indicates an rms difference of 0.41 A. The PEP in the KDOPS.PEP complex is anchored to the enzyme in a conformation that blocks its si face and leaves its re face largely devoid of contacts. This results from KDOPS's selective choice of a PEP conformer in which the phosphate group of PEP is extended toward the si face. Furthermore, the structure reveals that the bridging (P-O-C) oxygen atom and the carboxylate group of PEP are not strongly hydrogen-bonded to the enzyme. The resulting high degree of negative charge on the carboxylate group of PEP would then suggest that the condensation step between PEP and D-arabinose-5-phosphate (A5P) should proceed in a stepwise fashion through the intermediacy of a transient oxocarbenium ion at C2 of PEP. The molecular structural results are discussed in light of the chemically similar but mechanistically distinct reaction that is catalyzed by the enzyme 3-deoxy-D-arabino-2-heptulosonate-7-phosphate synthase and in light of the preferred enzyme-bound states of the substrate A5P.

Stereochemistry of family 52 glycosyl hydrolases: a beta-xylosidase from Bacillus stearothermophilus T-6 is a retaining enzyme.

A beta-xylosidase from Bacillus stearothermophilus T-6 assigned to the uncharacterized glycosyl hydrolase family 52 was cloned, overexpressed in Escherichia coli and purified. The enzyme showed maximum activity at 65 degrees C and pH 5.6-6.3. The stereochemistry of the hydrolysis of p-nitrophenyl beta-D-xylopyranoside was followed by 1H-nuclear magnetic resonance. Time dependent spectrum analysis showed that the configuration of the anomeric carbon was retained, indicating that a retaining mechanism prevails in family 52 glycosyl hydrolases. Sequence alignment and site-directed mutagenesis enabled the identification of functionally important amino acid residues of which Glu337 and Glu413 are likely to be the two key catalytic residues involved in enzyme catalysis.

Glutamic acid 160 is the acid-base catalyst of beta-xylosidase from Bacillus stearothermophilus T-6: a family 39 glycoside hydrolase.

A beta-xylosidase from Bacillus stearothermophilus T-6 was cloned, overexpressed in Escherichia coli and purified to homogeneity. Based on sequence alignment, the enzyme belongs to family 39 glycoside hydrolases, which itself forms part of the wider GH-A clan. The conserved Glu160 was proposed as the acid-base catalyst. An E160A mutant was constructed and subjected to steady state and pre-steady state kinetic analysis together with azide rescue and pH activity profiles. The observed results support the assignment of Glu160 as the acid-base catalytic residue.

Overproduction and characterization of seleno-methionine xylanase T-6.

The extracellular xylanase from Bacillus stearothermophilus T-6 is a thermostable alkaline tolerant enzyme that was found to bleach pulp optimally at pH 9 and 65 degrees C, and was successfully used in a large-scale bio-bleaching mill trial. In an attempt to obtain a heavy atom derivative suitable for complete X-ray analysis, xylanase T-6 was labeled biosynthetically with seleno-methionine, resulting in a 'built-in' array of atoms with specific X-ray anomalous scattering signal. Optimization of growth conditions resulted in over 0.8 g of homogeneous seleno-methionine xylanase T-6 per liter culture. The seleno-methionine enzyme was shown to be fully active and produced single crystals suitable for complete multiple wavelength anomalous diffraction (MAD) structural analysis.

Towards the development of novel antibiotics: synthesis and evaluation of a mechanism-based inhibitor of Kdo8P synthase.

The design and two synthetic pathways to aminophosphonate 4 which mimics the ionic and steric properties of putative oxocarbenium intermediate 3 in the Kdo8P synthase-catalyzed reaction are reported. It was found that 4 is a slow-binding, most potent inhibitor of the enzyme yet tested, with a Ki value of 0.4 microM.

[Organofluorine derivatives of amphotericin B: synthesis and antifungal activity]. / Ftororganicheskie poizvodnye amfoteritsina B: sintez i protivogribkovaia aktivnost'.

Reactions of amphotericin B, a polyene macrolide antibiotic, with acyl perfluorides resulted in formation of its N-perfluoroacyl derivatives. Physicochemical and medicobiological properties of the derivatives were studied. The biological study revealed that the acute toxity (LD50) of the derivatives was 2 times as low as that of the starting antibiotic. The derivatives showed high antifungal activity against a great number of the test cultures.

[The possibility of using the mycelial wastes from the production of antifungal antibiotics as additives to lubricating oils]. / Izuchenie vozmozhnosti ispol'zovaniia mitselial'nykh otkhodov proizvodstva protivogribkovykh antibiotikov v kachestve prisadok k smazochnym maslam.

Antiwear and antitear properties of mycelial waste from production of antifungal antibiotics i.e. levorin, nystatin, mycoheptin, amphotericin B and griseofulvin were studied. It was shown that the waste mycelium from griseofulvin production had the best antiwear and antitear characteristics due to a higher percentage of phosphorus and sulphur in it as compared to the mycelial waste from production of the other antibiotics.

[Ionol phosphorus organic analogs and stabilization of levorin]. / Ispol'zovaniephosphoroorganicheskikh analogov ionola dlia stabilizatsii levorina.

Stability of levorin isolated and purified with the use of ionole phosphorus organic analogs having in their structure phosphate, phosphonate and phosphonite groups was studied. The compound having in its structure (in addition to tertiary butyl substitutes) the phosphonite group with the P-H bond increasing the compound antioxidant property was shown to have the highest stabilizing effect. The results of the study made it possible to recommend the use of the space complicated phenols with the structure fragments of the P-H bond type as antioxidants in production of levorin.

[Fungicidal activity of imbricin and its salts]. / Fungitsidnaia aktivnost' imbritsina i ego solei.

The fungicidal activity of imbricin and its salts against fungal phytopathogens was studied. The biological tests revealed that the compounds had a fungicidal activity and inhibited the development of a number of plant diseases. Imbricin showed a marked fungicidal activity when used as a disinfectant of wheat seeds and in the control of root rot of cucumbers. It was demonstrated that imbricin and its salts had the growth regulating activity in plant cell culture.

[Synthesis and biological activity of hydrophosphoryl derivatives of amphotericin B]. / Sintez i biologicheskaia aktivnost' gidrofosforil'nykh proizvodnykh amfoteritsina b.

Reactions of amphotericin B, a polyenic macrolide antibiotic, with aromatic aldehydes and H3PO2 resulted in formation of its new hydrophosphoryl derivatives. Physico-chemical and biological properties of the compounds were investigated. The biological studies showed that the hydrophosphoryl derivatives of amphotericin B were low toxic and had antifungal and antiviral activities.

Catalytic mechanism of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase. The use of synthetic analogues to probe the structure of the putative reaction intermediate.

The proposed mechanistic pathway for the reaction catalyzed by 3-deoxy-D-manno-2-octulosonate-8-phosphate (Kdo8P) synthase was examined in terms of the structure of the putative bisphosphate intermediate. Two 2-deoxy analogues of the product Kdo8P, having been structurally prohibited from undergoing the ring-opening and possessing the stereochemistry of either the alpha-pyranase (compound 1) or the beta-pyranose form (compound 2) of the product, were synthesized and probed as inhibitors for the synthase. It was found that both analogues bind to the enzyme and are competitive inhibitors with respect to phosphoenolpyruvate binding, having Ki values of 470 microM and 303 microM, respectively. Comparison of this data to the Ki value of the tautomeric mixture of the product Kdo8P (Ki = 590 microM) suggests that both the alpha- and the beta-pyranose anomers (65.8% and 3.1%, respectively at neutral pH) bind to the enzyme with a slight (1.13 kJ/mol) preference for the beta-anomer, and that the C2 hydroxyl does not contribute to the binding. This uncertain stereochemical preference exhibited by the enzyme for the stereoisomers at the anomeric carbon suggests that the carboxylate binding site of the product is indistinct, while the hydroxyl and carboxylate binding sites may be interchangeable. More importantly, however, the isosteric phosphonate analogue 2,6-anhydro-3-deoxy-2 beta-phosphonylmethyl-8-phosphate-D-glycero-D-talo-octonate (3), which mimics the topological and electrostatic properties of the proposed cyclic intermediate, was found to be the most potent inhibitor of the enzyme with a Ki value of 5 microM. Two hitherto unrecognized aspects of the mechanism of the synthase were identified. First, the results showing that the cyclic analogues 1, 2 and 3 are inhibitors of the enzyme whereas the previously reported acyclic analogue, which contains no carbonyl group at C2 and may thus resemble the open-chain form of Kdo8P, is not an inhibitor, suggest that the pyranose form and not the open-chain acyclic form of the putative bisphosphate intermediate is handled by the enzyme. Second, since the overall stereochemical course of the transformation mediated by the synthase has been shown to involve si face addition of phosphoenolpyruvate to the re face of the carbonyl of arabinose 5-phosphate, the present observation involving analogue 3 suggest that the bisphosphate intermediate formed during the initial steps of synthesis may have the pyranose structure with the anomeric phosphate located in the beta-configuration.

[Comparative study of fungicidal and herbicidal activities of salts of polyenic macrolide antibiotics]. / Sravnitel'noe izuchenie fungitsidnoi i gerbitsidnoi aktivnosti solei polienovykh makrolidnykh antibiotikov.

The fungicidal and herbicidal activities of water soluble salts of levorin, nystatin, mycoheptin and amphotericin B, polyenic macrolide antibiotics, were studied. The biological tests showed that the antibiotics had fungicidal and low herbicidal activities. They also had a growth regulating action.

[Stabilization of standard samples of levorin and nystatin with antioxidants]. / Stabilizatsiia standartnykh obraztsov levorina i nistatina antioksidantami.

To prolong the storage period of the reference samples of levorin and nystatin, polyenic macrolide antibiotics, their effect of 23 antioxidants was studied by using rapid inactivation. The antioxidants belonged to compounds of different classes. The inactivation was performed at 20, 37 and 50 degrees C in the presence of 1, 2 or 5 per cent of the antioxidants. An antioxidant of the class of partially hydrated oxyquinolines was shown to have the highest stabilizing action on levorin and nystatin. It may be recommended for stabilizing the levorin and nystatin reference samples.

[Synthesis and biological activity of aminophenylphosphonic derivatives of levorin]. / Poluchenie i biologicheskaia aktivnost' aminofenilfosfonistykh proizvodnykh levorina.

Reactions of levorin, a polyenic macrolide antibiotic, with aromatic aldehydes and hypophosphorous acid resulted in formation of its amino phenylphosphonium derivatives. Physicochemical and biological properties of the derivatives were studied. The levorin amino phenylphosphonium derivatives were shown to be low toxic and have antifungal and antiviral activities.