Lipase-Catalyzed Regioselective Acylation and Deacylation of Glucose Derivatives

In the development of an efficient synthesis of 1-O-decanoyl-2,3,4,6-tetra-O-acetyl-β-D-glucose (β-2) several lipase-based approaches have been explored. Among five immobilized Upases tested, the lipase from Candida antarctica proved particularly efficient for catalyzing selective hydrolysis in the 1-position of 1,2,3,4,6-penta-O-acetyl-β-D-glucose (β-1). Using triethylamine as catalyst, the hydrolysis product 2,3,4,6-tetra-O-acetyl-D-glucose (3) can be esterified with decanoyl chloride to form β-2 selectively, thereby providing an efficient chemo-enzymatic synthesis starting from readily available raw materials. Attempts to produce β-2 from β-1 by lipase-catalyzed interesterification or to esterify 3 with decanoic acid using a lipase as catalyst were unsuccessful. The latter finding was explained by the hemiacetal OH group of glucose being unable to act as nucleophile in the lysis of the lipase acyl-enzyme intermediate. Furthermore, β-2 was found to bee a too bulky substrate to fit into the active site of any of the lipases tested.     

Antiviral compounds from traditional Chinese medicines Galla Chinese as inhibitors of HCV NS3 protease

Under the guidance of bioassay, the EtOAc extract fraction of the Traditional Chinese Medicine (TCM) Galla Chinese was found to be efficient in inhibiting the NS3 protease of HCV and purified the fraction to get three polyphenol compounds 1,2,6-tri-O-galloyl-β-D-glucose (1), 1,2,3,6-tetra-O-galloyl-β-D-glucose (2), and 1,2,3,4,6-penta-O-galloyl-β-D-glucose (3), which were identified as inhibitors of Hepatitis C Virus (HCV) NS3 protease. Compounds 1, 2, and 3 inhibited HCV NS3 protease with IC₅₀ of 1.89, 0.75, and 1.60 μM, respectively.     

Effect of Naturally Occurring Flavonoids on Lipid Peroxidation and Membrane Permeability Transition in Mitochondria

The ability of eight structurally related naturally occurring flavonoids in inhibiting lipid peroxidation and mitochondrial membrane permeability transition (MMPT), as well as respiration and protein sulfhydryl oxidation in rat liver mitochondria, was evaluated. The flavonoids tested exhibited the following order of potency to inhibit ADP/Fe(II)-induced lipid peroxidation, estimated with the thiobarbituric acid assay: 3′-O-methyl-quercetin > quercetin > 3,5,7,3′,4′-penta-O-methyl-quercetin > 3,7,3′,4′-tetra-O-methyl-quercetin > pinobanksin > 7-O-methyl-pinocembrin > pinocembrin > 3-O-acyl-pinobanksin. MMPT was estimated by the extent of mitochondrial swelling induced by 10 μM CaCl₂ plus 1.5 mM inorganic phosphate or 30 μM mefenamic acid. The most potent inhibitors of MMPT were quercetin, 7-O-methyl-pinocembrin, pinocembrin, and 3,5,7,3′,4′-penta-O-methyl-quercetin. The first two inhibited in parallel the oxidation of mitochondrial protein sulfhydryl involved in the MMPT mechanism. The most potent inhibitors of mitochondrial respiration were 7-O-methyl-pinocembrin, quercetin, and 3′-O-methyl-quercetin while the most potent uncouplers were pinocembrin and 3-O-acyl-pinobanksin. In contrast 3,7,3′,4′-tetra-O-methyl-quercetin and 3,5,7,3′,4′-penta-O-methyl-quercetin showed the lowest ability to affect mitochondrial respiration. We conclude that, in general, the flavonoids tested are able to inhibit lipid peroxidation on the mitochondrial membrane and/or MMPT. Multiple methylation of the hydroxyl substitutions, in addition to sustaining good anti-lipoperoxidant activity, reduces the effect of flavonoids on mitochondrial respiration, and therefore, increases the pharmacological potential of these compounds against pathological processes related to oxidative stress.     

Immobilization of thermostable β-glucosidase from Sulfolobus shibatae by cross-linking with transglutaminase

Thermostable β-glucosidase from Sulfolobus shibatae was immobilized on silica gel modified or not modified with 3-aminopropyl-triethoxysilane using transglutaminase as a cross-linking factor. Obtained preparations had specific activity of 3883 U/g of the support, when measured at 70 °C using o-nitrophenyl β-d-galactopyranoside (GalβoNp) as substrate. The highest immobilization yield of the enzyme was achieved at pH 5.0 in reaction media. The most active preparations of immobilized β-glucosidase were obtained at a transglutaminase concentration of 40 mg/ml at 50 °C. The immobilization was almost completely terminated after 100 min of the reaction and prolonged time of this process did not cause considerable changes of the activity of the preparations. The immobilization did not influence considerably on optimum pH and temperature of GalβoNp hydrolysis catalyzed by the investigated enzyme (98 °C, pH 5.5). The broad substrate specifity and properties of the thermostable β-glucosidase from S. shibatae immobilized on silica-gel indicate its suitability for hydrolysis of lactose during whey processing.     

The interaction between lactose level and enzyme supplementation and form of barley processing on performance, digestibility and faecal volatile fatty acid concentration of weanling pigs fed barley-based diets

A 2×3 factorial arrangement was used to investigate the interaction between lactose level (170 g/kg versus 275 g/kg), enzyme supplementation (with or without endo-1,3 (4)-β-glucanase) and toasting (raw barley versus toasted barley) in barley-based diets (250 g/kg) on piglet performance and diet digestibility postweaning. One hundred and forty-four weaned piglets (24 days old, 6.5 kg live weight) were blocked on the basis of live weight and were assigned to one of six dietary treatments (n = 6) for 21 days as follows: (1) 170 g lactose/kg and barley (250 g/kg), (2) 275 g lactose/kg and barley (250 g/kg), (3) 170 g lactose/kg and toasted-flaked barley (250 g/kg), (4) 275 g lactose/kg and toasted-flaked barley (250 g/kg), (5) 170 g lactose/kg and barley (250 g/kg) plus β-glucanase, (6) 275 g lactose/kg and barley (250 g/kg) plus β-glucanase. There was an interaction (P<0.05) between lactose level and β-glucanase supplementation on average daily gain (ADG), food conversion ratio (FCR) and coefficient of total tract apparent digestibilities (CTTAD) of dry matter (DMD), organic matter (OMD) and nitrogen (N). Pigs offered 170 g lactose/kg and β-glucanase supplementation had an improved FCR and ADG compared to the treatment based on 170 g lactose/kg and unsupplemented barley. However, β-glucanase supplementation at 275 g lactose/kg had no significant effect on FCR and ADG compared to the 275 g lactose/kg and unsupplemented barley. Pigs offered diets containing 170 g lactose/kg plus β-glucanase had a significantly higher CTTAD for DMD, OMD and nitrogen compared to the 275 g lactose/kg plus β-glucanase diet. However, there was no significant effect of lactose level in the unsupplemented diets. Pigs offered high lactose diets showed significantly increased total VFA concentration, molar proportions of butyric acid and reduced acetic acid compared to those offered low lactose diets. Pigs offered β-glucanase supplemented diets showed reduced (P<0.05) total VFA concentration compared to unsupplemented diets. In conclusion, β-glucanase supplementation improved diet digestibility and pig performance compared to the unsupplemented diet at the low lactose level only. There was no response of β-glucanase supplementation at the high level of lactose.     

Chiral macrocyclic compounds from lactose derivatives

The reaction of benzyl 2,6,6′-tri-O-benzyl-3′,4′-O-isopropylidene-β-lactoside with 1,11-ditosyloxy-3,6,9-trioxaundecane gave benzyl 2,6,6′-tri-O-benzyl-3′,4′-O-isopropylidene-3,2′-O--(3,6,9-trioxaundecane-1,11-diyl)-β-lactoside (2, 47%). Acid hydrolysis of 2 and condensation of the product with 1,14-ditosyloxy-3,6,9,12-tetra-oxatetradecane afforded benzyl 2,6,6′-tri-O-benzyl-3′,4′-O-(3,6,9,12-tetraoxa-tetradecane-1,14-diyl)-3,2′-O-(3,6,9-trioxaundecane-1,11-diyl)-β-lactoside (29%). Similarly, the reaction of benzyl 2,6,2′,4′,6′-penta-O-benzyl-β-lactoside with Ts[OCH₂CH₂]₄OTs gave benzyl 2,6,2′,4′,6′-penta-O-benzyl-3,3′-O-(3,6,9-trioxaundecane-1,11-diyl)-β-lactoside (78%). ¹H-N.m.r. spectroscopy has been used to study the formation of host-guest complexes with some of these macrocyclic compounds and benzyl ammonium thiocyanate.     

Streptococcus pneumoniae type XIV polysaccharide: synthesis of a repeating branched tetrasaccharide with dioxa-type spacer-arms

β-Glycosides of 2-acetamido-2-deoxy- -glucopyranose were synthesized, using either 7-methoxycarbonyl-3,6-dioxa-1-heptanol or 8-azido-3,6-dioxa-1-octanol. Selective β-lactosylation of 7-methoxycarbonyl-3,6-dioxaheptyl 2-acetamido-3-O-benzyl-2-deoxy-β- -glucopyranoside with hepta-O-acetyl-lactosyl-trichloroacetimidate, followed by β-galactosylation of the secondary hydroxyl group with O-(2,3,4,6-tetra-O-acetyl-- -galactopyranosyl)trichloroacetimidate, catalytic hydrogenolysis, and O-deacetylation, gave 7-methoxycarbonyl-3,6-dioxaheptyl 2-acetamido-2-deoxy-4-O-β- -galactopyranosyl-6-O-(4-O-β- -galactopyranosyl-β- -glucopyranosyl)β- -glucopyranoside. Selective β-lactosylation of 8-azido-3,6-dioxaocytl 2-acetamido-3-O-benzyl-2-deoxy-β- -glucopyranoside with hepta-O-acetyl-lactosyl bromide in the presence of silver triflate, followed by condensation with 2,3,4,6-tetra-O-acetyl-- -galactopyranosyl bromide in the presence of silver triflate, catalytic hdyrogenolysis, and O-deacetylation, gave 8-azido-3,6-dioxaoctyl 2-acetamido-2-deoxy-4-O-β- -galactopyranosyl-6-O-(4-O-β- -galactopyranosyl-β- -glucopyranosyl)-β- glucopyranoside.     

Screening of lipases for regioselective hydrolysis of peracetylated β-monosaccharides

The monodeacetylation of peracetylated-β-d-galactose (1) and peracetylated N-acetyl-β-d-glucosamine (2) by different lipases is here described. Lipases from different sources in an immobilized form were evaluated to find those that offer the higher activity and regioselectivity in the reactions. In the hydrolysis of 1, the lipase from Aspergillus niger was the most active one, although it hydrolyzed the anomeric position. Using the lipase from Candida rugosa, 30% yield of the corresponding 6-OH isomer was achieved. On the other hand, in the hydrolysis of 2, the lipase from A. niger was the most active and regioselective catalyst, producing more than 75% of the 6-OH derivative product.     

Identification and Characterization of γ-Glutamylamine Cyclotransferase,an Enzyme Responsible for γ-Glutamyl-?-lysine Catabolism

γ-Glutamylamine cyclotransferase (GGACT) is an enzyme that converts γ-glutamylamines to free amines and 5-oxoproline. GGACT shows high activity toward γ-glutamyl-ϵ-lysine, derived from the breakdown of fibrin and other proteins cross-linked by transglutaminases. The enzyme adopts the newly identified cyclotransferase fold, observed in γ-glutamylcyclotransferase (GGCT), an enzyme with activity toward γ-glutamyl-α-amino acids (Oakley, A. J., Yamada, T., Liu, D., Coggan, M., Clark, A. G., and Board, P. G. (2008) J. Biol. Chem. 283, 22031–22042). Despite the absence of significant sequence identity, several residues are conserved in the active sites of GGCT and GGACT, including a putative catalytic acid/base residue (GGACT Glu⁸²). The structure of GGACT in complex with the reaction product 5-oxoproline provides evidence for a common catalytic mechanism in both enzymes. The proposed mechanism, combined with the three-dimensional structures, also explains the different substrate specificities of these enzymes. Despite significant sequence divergence, there are at least three subfamilies in prokaryotes and eukaryotes that have conserved the GGCT fold and GGCT enzymatic activity.     

Synthesis and structural study of 5-nitro-6-(penta-O-acetylpentitol-1-yl)norbornenes

Uncatalyzed reaction between cyclopentadiene and (E)-3,4,5,6,7-pentaacetoxy-1-nitrohept-1-enes having the -manno, -galacto, and -gluco configurations at C-3—C-7 led, in each case, to the four stereoisomeric 5-nitro-6-(1,2,3,4,5-penta-O-acetylpentitol-1-yl)bicyclo[2.2.1]hept-2-enes. Face selectivity is discussed in terms of the sugar-chain configuration. The structures assigned the adducts are based on their n.m.r. spectra, and, in the case of the -manno compounds, on X-ray data. Also described are the ¹³C-n.m.r. spectra of the starting nitroalkenes. The crystal structures of (5S,6S)1,2,3,4,5-penta-O-acetyl-1-C-(5-exo-nitrobicyclo[2.2.1]hept-2-en-6-endo-yl- -manno-pentitol (3a) and (5S,6S)1,2,3,4,5-penta-O-acetyl-1-C-(5-endo-nitrobicyclo[2.2.1]hept-2-en-6-exo-yl- -manno-pentitol (5a) were determined from three-dimensional, X-ray data. Crystals of 3a are monoclinic, space group P2₁, with two molecules in a cell of dimensions a = 9.054(3), b = 15.580(11), c = 10.138(4) Å, β = 116.27(3)°. The structure was refined to an R-factor of 0.050 on the basis of 1485 observations. Crystals of 5a are triclinic, space group P1, with one molecule in a cell of dimensions a = 8.680(4), b = 9.760(4), c = 8.695(7) Å, = 98.69(5), β = 103.13(5), γ = 112.09(3)°. The structure was refined to an R-factor of 0.074 based on 970 observations.     

Equilibrium shifts in enzyme reactions at high pressure

The effect of pressure on the equilibrium of a reaction was studied. Theoretical equilibrium constants and product concentrations have been calculated at elevated pressures. The theory is illustrated with an example of l-malate synthesis catalyzed by a fumarase. To study shifts in the equilibrium relatively low pressures can be applied (50–200 MPa), but our calculations show that for process optimisation much higher pressures (up to 1000 MPa) have to be used.

At these higher pressures, more stable enzymes are needed. We performed experiments with the hyperthermophilic β-glycosidase from Pyrococcus furiosus as a catalyst. Oligosaccharides were synthesized from glucose in an equilibrium reaction at pressures from 0.1 to 500 MPa. The enzyme remained active at 500 MPa. The equilibrium of the reaction was influenced by pressure and shifted towards the hydrolysis side, decreasing final oligosaccharide concentrations with increasing pressure. This pressure dependence of the final product concentration and the equilibrium constant could be described with a positive reaction volume of 2.4 mol/cm³.     

A novel enzyme immunoassay based on potentiometric measurement of molecular adsorption events by an extended-gate field-effect transistor sensor

We developed a novel enzyme immunoassay based on a potentiometric measurement of molecular adsorption events by using an extended-gate field-effect transistor (FET) sensor. The adsorbing rate of a thiol compound on a gold surface was found to depend on the concentration of the compound. To construct an electrochemical enzyme immunoassay system by using the sensor, the enzyme chemistry of acetylcholinesterase (AChE) to generate a thiol compound was used and combined with the enzyme-linked immunosorbent assays (ELISA). After the AChE-catalyzed reaction, the amount of the antigen was obtained by detecting the adsorbing rate of the generated thiol compound on the gold electrode using the FET sensor. The measurement stability was also found to improve when a high frequency voltage of 10 kHz or more was superimposed to the reference electrode. The signal corresponding to a range between 1 and 250 pg/mL of Interleukin 1β was obtained by the FET sensor when a voltage of 1 MHz was superimposed onto the reference electrode. The FET sensor based ELISA used in this measurement technique can successfully detect Interleukin 1β at concentrations as low as 1 pg/mL.     

Vinyl imidazole carrying metal-chelated beads for reversible use in yeast invertase adsorption

Poly(ethylene glycol dimethacrylate-n-vinyl imidazole) [poly(EGDMA–VIM)] hydrogel (average diameter 150–200 μm) was prepared copolymerizing ethylene glycol dimethacrylate (EGDMA) with n-vinyl imidazole (VIM). Poly(EGDMA–VIM) beads had a specific surface area of 59.8 m²/g. Poly(EGDMA–VIM) beads were characterized by swelling studies and scanning electron microscope (SEM). Cu²⁺ ions were chelated on the poly(EGDMA–VIM) beads (452 μmol Cu²⁺/g), then the metal-chelated beads were used in the adsorption of yeast invertase in a batch system. The maximum invertase adsorption capacity of the poly(EGDMA–VIM)–Cu²⁺ beads was observed as 35.2 mg/g at pH 4.5. The adsorption isotherm of the poly(EGDMA–VIM)–Cu²⁺ beads can be well fitted to the Langmuir model. Adsorption kinetics data were tested using pseudo-first- and -second-order models. Kinetic studies showed that the adsorption followed a pseudo-second-order reaction. The value of the Michaelis constant K_m of invertase was significantly larger upon adsorption, indicating decreased affinity by the enzyme for its substrate, whereas V_max was smaller for the adsorbed invertase. The optimum temperature for the adsorbed preparation of poly(EGDMA–VIM)–Cu²⁺-invertase at 50 °C, 10 °C higher than that of the free enzyme at 40 °C. Storage stability was found to increase with adsorption. Adsorbed invertase retains an activity of 82% after 10 batch successive reactions, demonstrating the usefulness of the enzyme-loaded beads in biocatalytic applications.     

Structural and biochemical characterization of the salicylyl-acyltranferase SsfX3 from a tetracycline biosynthetic pathway

SsfX3 is a GDSL family acyltransferase that transfers salicylate to the C-4 hydroxyl of a tetracycline intermediate in the penultimate step during biosynthesis of the anticancer natural product SF2575. The C-4 salicylate takes the place of the more common C-4 dimethylamine functionality, making SsfX3 the first acyltransferase identified to act on a tetracycline substrate. The crystal structure of SsfX3 was determined at 2.5 Å, revealing two distinct domains as follows: an N-terminal β-sandwich domain that resembles a carbohydrate-binding module, and a C-terminal catalytic domain that contains the atypical α/β-hydrolase fold found in the GDSL hydrolase family of enzymes. The active site lies at one end of a large open binding pocket, which is spatially defined by structural elements from both the N- and C-terminal domains. Mutational analysis in the putative substrate binding pocket identified residues from both domains that are important for binding the acyl donor and acceptor. Furthermore, removal of the N-terminal carbohydrate-binding module-like domain rendered the stand-alone α/β-hydrolase domain inactive. The additional noncatalytic module is therefore proposed to be required to define the binding pocket and provide sufficient interactions with the spatially extended tetracyclic substrate. SsfX3 was also demonstrated to accept a variety of non-native acyl groups. This relaxed substrate specificity toward the acyl donor allowed the chemoenzymatic biosynthesis of C-4-modified analogs of the immediate precursor to the bioactive SF2575; these were used to assay the structure activity relationships at the C-4 position.     

Temperature and carbon source affect the production and secretion of a thermostable β-xylosidase by Aspergillus fumigatus

The effect of the temperature of growth and carbon source on the production and secretion of β-xylosidase (EC 3.2.1.37) by the thermotolerant fungi Aspergillus fumigatus was studied in submerged cultures. In cultures developed at optimal temperature (30 °C), the enzyme was predominantly cell-bound, while in cultures developed at higher temperature (42 °C), the β-xylosidase activity was predominantly found in the cell-free filtrates. The use of corn cob powder instead of xylan as substrate increased considerably the secretion of enzyme. The highest level of extracellular β-xylosidase (45 U/ml or 360 U/mg protein) was obtained in 3% corn cob cultures grown at 42 °C for 72 h. The partially purified enzyme was active and stable at high temperatures. The presence of high titres of β-xylosidase activity in association with xylanase in the culture filtrates enhanced the efficiency of the pulp hydrolysis process.     

Structure and Catalytic Mechanism of 3-Ketosteroid-{Delta}4-(5α)-dehydrogenase from Rhodococcus jostii RHA1 Genome

3-Ketosteroid Δ4-(5α)-dehydrogenases (Δ4-(5α)-KSTDs) are enzymes that introduce a double bond between the C4 and C5 atoms of 3-keto-(5α)-steroids. Here we show that the ro05698 gene from Rhodococcus jostii RHA1 codes for a flavoprotein with Δ4-(5α)-KSTD activity. The 1.6 Å resolution crystal structure of the enzyme revealed three conserved residues (Tyr-319, Tyr-466, and Ser-468) in a pocket near the isoalloxazine ring system of the FAD co-factor. Site-directed mutagenesis of these residues confirmed that they are absolutely essential for catalytic activity. A crystal structure with bound product 4-androstene-3,17-dione showed that Ser-468 is in a position in which it can serve as the base abstracting the 4β-proton from the C4 atom of the substrate. Ser-468 is assisted by Tyr-319, which possibly is involved in shuttling the proton to the solvent. Tyr-466 is at hydrogen bonding distance to the C3 oxygen atom of the substrate and can stabilize the keto-enol intermediate occurring during the reaction. Finally, the FAD N5 atom is in a position to be able to abstract the 5α-hydrogen of the substrate as a hydride ion. These features fully explain the reaction catalyzed by Δ4-(5α)-KSTDs.     

A Triple Mutant in the Ω-loop of TEM-1 β-Lactamase Changes the Substrate Profile via a Large Conformational Change and an Altered General Base for Catalysis

β-Lactamases are bacterial enzymes that hydrolyze β-lactam antibiotics. TEM-1 is a prevalent plasmid-encoded β-lactamase in Gram-negative bacteria that efficiently catalyzes the hydrolysis of penicillins and early cephalosporins but not oxyimino-cephalosporins. A previous random mutagenesis study identified a W165Y/E166Y/P167G triple mutant that displays greatly altered substrate specificity with increased activity for the oxyimino-cephalosporin, ceftazidime, and decreased activity toward all other β-lactams tested. Surprisingly, this mutant lacks the conserved Glu-166 residue critical for enzyme function. Ceftazidime contains a large, bulky side chain that does not fit optimally in the wild-type TEM-1 active site. Therefore, it was hypothesized that the substitutions in the mutant expand the binding site in the enzyme. To investigate structural changes and address whether there is an enlargement in the active site, the crystal structure of the triple mutant was solved to 1.44 Å. The structure reveals a large conformational change of the active site Ω-loop structure to create additional space for the ceftazidime side chain. The position of the hydroxyl group of Tyr-166 and an observed shift in the pH profile of the triple mutant suggests that Tyr-166 participates in the hydrolytic mechanism of the enzyme. These findings indicate that the highly conserved Glu-166 residue can be substituted in the mechanism of serine β-lactamases. The results reveal that the robustness of the overall β-lactamase fold coupled with the plasticity of an active site loop facilitates the evolution of enzyme specificity and mechanism.     

Six new C21 steroidal glycosides from Asclepias curassavica L

Six new C₂₁ steroidal glycosides, named curassavosides A–F (3–8), were obtained from the aerial parts of Asclepias curassavica (Asclepiadaceae), along with two known oxypregnanes, 12-O-benzoyldeacylmetaplexigenin (1) and 12-O-benzoylsarcostin (2). By spectroscopic methods, the structures of the six new compounds were determined as 12-O-benzoyldeacylmetaplexigenin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (3), 12-O-benzoylsarcostin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (4), sarcostin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (5), sarcostin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-digitoxopyranoside (6), 12-O-benzoyldeacylmetaplexigenin 3-O-β-d-glucopyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (7), and 12-O-benzoylsarcostin 3-O-β-d-glucopyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (8), respectively. All compounds (1–8) were tested for in vitro cytotoxicity; only compound 3 showed weak inhibitory activity against Raji and AGZY cell lines.     

Production, purification and properties of γ-glutamyltranspeptidase from a newly isolated Bacillus subtilis NX-2

Production, purification and properties of γ-glutamyltranspeptidase from a newly isolated Bacillus subtilis NX-2 was investigated. At the optimum conditions for enzyme formation, a high level, 3.2 U/ml of γ-GTP was obtained. The extracellular γ-GTP from this strain was purified 111.15-fold to homogeneity from the culture supernatant by acetone precipitation, hydrophobic interaction chromatography and ion exchange chromatography. The purified enzyme was a heterodimer consisting of one large subunit (43 kDa) and one small subunit (32 kDa), and exhibited high activity at 40–60 °C, pH 8.0. It preferred basic amino acids as γ-glutamyl acceptor in transpeptidation, and the stereochemistry of the γ-glutamyl acceptor had no influence on the enzyme activity, which was different from other γ-GTPs reported. Furthermore, it was proved that γ-GTP of this strain could catalyze the transfer of l-glutamine to glycylglycine to synthesize Gln–Gly–Gly, which was promising for the synthesis of valuable γ-glutamyl peptides.     

Biochemical and structural studies of uncharacterized protein PA0743 from Pseudomonas aeruginosa revealed NAD+-dependent L-serine dehydrogenase

The β-hydroxyacid dehydrogenases form a large family of ubiquitous enzymes that catalyze oxidation of various β-hydroxy acid substrates to corresponding semialdehydes. Several known enzymes include β-hydroxyisobutyrate dehydrogenase, 6-phosphogluconate dehydrogenase, 2-(hydroxymethyl)glutarate dehydrogenase, and phenylserine dehydrogenase, but the vast majority of β-hydroxyacid dehydrogenases remain uncharacterized. Here, we demonstrate that the predicted β-hydroxyisobutyrate dehydrogenase PA0743 from Pseudomonas aeruginosa catalyzes an NAD⁺-dependent oxidation of l-serine and methyl-l-serine but exhibits low activity against β-hydroxyisobutyrate. Two crystal structures of PA0743 were solved at 2.2–2.3-Å resolution and revealed an N-terminal Rossmann fold domain connected by a long α-helix to the C-terminal all-α domain. The PA0743 apostructure showed the presence of additional density modeled as HEPES bound in the interdomain cleft close to the predicted catalytic Lys-171, revealing the molecular details of the PA0743 substrate-binding site. The structure of the PA0743-NAD⁺ complex demonstrated that the opposite side of the enzyme active site accommodates the cofactor, which is also bound near Lys-171. Site-directed mutagenesis of PA0743 emphasized the critical role of four amino acid residues in catalysis including the primary catalytic residue Lys-171. Our results provide further insight into the molecular mechanisms of substrate selectivity and activity of β-hydroxyacid dehydrogenases.