Properties of 2,3-Butanediol Dehydrogenases from Lactococcus lactis subsp. lactis in Relation to Citrate Fermentation

Two 2,3-butanediol dehydrogenases (enzymes 1 and 2; molecular weight of each, 170,000) have been partially purified from Lactococcus lactis subsp. lactis (Streptococcus diacetylactis) D10 and shown to have reductase activity with either diacetyl or acetoin as the substrate. However, the reductase activity with 10 mM diacetyl was far greater for both enzymes (7.0- and 4.7-fold for enzymes 1 and 2, respectively) than with 10 mM acetoin as the substrate. In contrast, when acetoin and diacetyl were present together, acetoin was the preferred substrate for both enzymes, with enzyme 1 showing the more marked preference for acetoin. meso-2,3-Butanediol was the only isomeric product, with enzyme 1 independent of the substrate combinations. For enzyme 2, both the meso and optical isomers of 2,3-butanediol were formed with acetoin as the substrate, but only the optical isomers were produced with diacetyl as the substrate. With batch cultures of strain D10 at or near the point of citrate exhaustion, the main isomers of 2,3-butanediol present were the optical forms. If the pH was sufficiently high (>pH 5), acetoin reduction occurred over time and was followed by diacetyl reduction, and meso-2,3-butanediol became the predominant isomer. Interconversion of the optical isomers into the meso isomer did occur. The properties of 2,3-butanediol dehydrogenases are consistent with diacetyl and acetoin removal and the appearance of the isomers of 2,3-butanediol.     

Cloning,expression and characterization of glycerol dehydrogenase involved in 2,3-butanediol formation in Serratia marcescens H30

The meso-2,3-butanediol dehydrogenase (meso-BDH) from S. marcescens H30 is responsible for converting acetoin into 2,3-butanediol during sugar fermentation. Inactivation of the meso-BDH encoded by budC gene does not completely abolish 2,3-butanediol production, which suggests that another similar enzyme involved in 2,3-butanediol formation exists in S. marcescens H30. In the present study, a glycerol dehydrogenase (GDH) encoded by gldA gene from S. marcescens H30 was expressed in Escherichia coli BL21(DE3), purified and characterized for its properties. In vitro conversion indicated that the purified GDH could catalyze the interconversion of (3S)-acetoin/meso-2,3-butanediol and (3R)-acetoin/(2R,3R)-2,3-butanediol. (2S,3S)-2,3-Butanediol was not a substrate for the GDH at all. Kinetic parameters of the GDH enzyme showed lower K _m value and higher catalytic efficiency for (3S/3R)-acetoin in comparison to those for (2R,3R)-2,3-butanediol and meso-2,3-butanediol, implying its physiological role in favor of 2,3-butanediol formation. Maximum activity for reduction of (3S/3R)-acetoin and oxidations of meso-2,3-butanediol and glycerol was observed at pH 8.0, while it was pH 7.0 for diacetyl reduction. The enzyme exhibited relative high thermotolerance with optimum temperature of 60 °C in the oxidation–reduction reactions. Over 60 % of maximum activity was retained at 70 °C. Additionally, the GDH activity was significantly enhanced for meso-2,3-BD oxidation in the presence of Fe²⁺ and for (3S/3R)-acetoin reduction in the presence of Mn²⁺, while several cations inhibited its activity, particularly Fe²⁺ and Fe³⁺ for (3S/3R)-acetoin reduction. The properties provided potential application for single configuration production of acetoin and 2,3-butanediol .     

Synthesis and anti-inflammatory activity of 2-oxo-2H-chromenyl and 2H-chromenyl-5-oxo-2,5-dihydrofuran-3-carboxylates

Cycloaddition reaction of 4-chloro-2-oxo-2H-chromene-3-carbaldehydes (3a-g) and 4-chloro-2H-chromene-3-carbaldehydes (7a-h) with activated alkynes (4a-b) provided the 2-oxo-2H-chromenyl-5-oxo-2,5-dihydrofuran-3-carboxylates (5a-n) and 2H-chromenyl-5-oxo-2,5-dihydrofuran-3-carboxylates (8a-p). All the prepared compounds were screened for anti-inflammatory activity. In vitro anti-inflammatory activity data demonstrated that the compounds 5g, 5i, 5k-l and 8f are effective among the tested compounds against TNF-α (1.108 ± 0.002, 0.423 ± 0.022, 0.047 ± 0.001, 0.070 ± 0.002 and 0.142 ± 0.001 µM) in comparison with standard compound Prednisolone (0.033 ± 0.002 µM). Based on in vitro results, three compounds (5i, 5k and 8f) have been selected for in vivo experiments and these compounds are identified as better compounds with respect to anti-inflammatory activity in LPS induced mice model. Compound 5i was identified as potent and showed significant reduction in TNF-α and IL-6.     

Uptake of exogenous metabolites by virus-transformed cells: changes induced by temperature and pH.

Bestatin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-(S)-leucine, inhibited aminopeptidase B and leucine aminopeptidase in a competitive manner and their K_i values were calculated to be 6 × 10^?8 and 2 × 10^?8M, respectively. Among all stereoisomers of bestatin synthesized, those which have a (2S)-configuration in the 3-amino-2-hydroxy-4-phenylbutanoyl moiety showed marked inhibition against aminopeptidase B and leucine aminopeptidase compared with the other isomers which have (2R)-configuration. One of the isomers, [(2S,3S)-3-amino-2-hydroxy-4-phenylbutanoyl]-(R)-leucine, showed somewhat stronger activity against aminopeptidase B than bestatin. Aminopeptidase B appears to be a metallo-exopeptidase. It is proposed that bestatin and its active isomers are effective due to a mechanism other than a chelating action at the active center.     

Four-coordinate complexes of bis(1-diphenylphosphinoindenyl)iron(II)

Palladium, platinum and rhodium complexes of rac- and meso-bis(1-diphenylphosphinoindenyl)iron(II) (1) are reported. Both rac and meso isomers of {bis(1-diphenylphosphinoindenyl)iron(II)}palladium dichloride (rac- and meso-2) were characterized by X-ray crystallography along with the rac isomer of the Pt analogue (rac-3). NMR analysis of the rhodium complex [{bis(1-diphenylphosphinoindenyl)iron(II)}(cyclooctadiene)rhodium(I)] tetraphenylborate suggests a similar structure in solution. Coupling reactions of n- and sec-BuCl with bromobenzene in THF are catalysed by rac-2 and found to be similar to (PPh₃)₂PdCl₂ but poorer than (dppf)PdCl₂ in diethyl ether.     

Biological activity of antheridic acid,an antheridiogen of anemia phyllitidis

The biological activities of synthetic antheridiogen of Anemia phyllitidis, (±)-antheridic acid, and naturally derived antheridic acid with regard to induction of antheridial formation and dark spore germination in A. phyllitidis were closely similar. The activity of (±)-3-epi-antheridic acid was weaker than that of (±)-antheridic acid in inducing these phenomena. (±)-Antheridic acid was active in inducing elongation growth in the dwarf rice bioassay system, although its activity was weaker than that of GA₃. In this bioassay system, (±)-3-epi-antheridic acid showed higher activity than did (±)-antheridic acid.     

Complexes of large ring macrocycles. The preparation of the C-meso- and C-racemic-diastereoisomers of Me6[18]aneN4 and characterisation of their ‘blue’ and ‘red’ copper(II) complexes

The reaction of 1,4-diaminobutane monohydroperchlorate with acetone gives trans-Me₆[18]- dieneN₄·2HClO₄·2H₂O. The diene can be reduced with NaBH₄ under basic conditions to give a mixture of C-meso Me₆[18]aneN₄ (melting point (m.p.) 119–120 °C) and C-racemic Me₆[18]aneN₄ (m.p. 79 °C) which can be separated by fractional crystallisation from xylene (Me₆[18]aneN₄=2,4,4,11,13, 13-hexamethyl-1,5,10,14-tetraazacyclooctadecane). The free base form of Me₆[18]dieneN₄ has been characterised and molecular weight measurements by vapour pressure osmometry confirm the 18-membered tetraaza structure rather than the alternative 9- membered diaza structure. Blue copper(II) complexes [CuL](ClO₄)₂ have been characterised with both C- meso and C-racemic Me₆[18] aneN₄ which have a dd band at 680 nm. These blue complexes are converted to the more thermodynamically stable red isomers (λ_max 488 nm) on stirring aqueous suspensions of the blue perchlorate salts. The red isomers are believed to have the trans III or RSSR configuration of the sec- NH centres.     

Occurrence of meso-alpha, epsilon-diaminopimelate dehydrogenase in Bacillus sphaericus.

A new amino acid dehydrogenase catalyzing the oxidative deamination of meso-α,?-diaminopimelate was found in the crude extract of Bacillus sphaericus IFO 3525. This dehydrogenase requiring NADP was specific for meso-diaminopimelate and the other isomers were not substrates. The enzyme was optimally active at about pH 10.5. NAD could not replace NADP.     

Studies on the Syntheses of the Pyrethrin Analogues and their Biological Activities

The separation of (±) -2,2-dimethyl-3- (3′,4′-methylenedioxyphenyl) -cyclopropane-1-carboxylic acid into the geometrical isomers and the assignment of their configurations were achieved. Of the two isomers, the (±) -trans-acid, which was found more toxic when esterified with (±) -allethrolone, was resolved by means of an optically active α-phenylethylamine salt into (+) - and (-) -enantiomers. (IR:3R) -Configuration was assigned to the (+) -trans-acid and (IS:3S) -configuration to the (-) -trans-acid. The bioassay revealed that the (±) -allethrolone ester with the (+) -trans-acid, which belongs to the same optical series as the natural chrysanthemum acids, was the most toxic against common houseflies, as was the case with other pyrethroids.     

Efficient synthesis of unnatural dipeptides based on cis-2,5-disubstituted pyrrolidine

The well-defined unnatural dipeptides based on cis-2,5-disubstituted pyrrolidine backbone were synthesized from commercially available starting materials meso-diethyl-2,5-dibromoadipate, (S)-(?)-1-phenylethylamine, and phenylalanine. The configurations of all the chiral centers in these unnatural dipeptides are confirmed by X-ray crystal diffraction analysis.     

Conversion of (2Z,4E)-5-(1′,2′-epoxy-2′,6′,6′-trimethylcyclohexyl)-3-methyl-2,4-pentadienoic acid to xanthoxin acid by Cercospora cruenta, a fungus producing (+)-abscisic acid

(±)-(2Z,4E)-5-(1′,2′-epoxy-2′,6′,6′-trimethylcyclohexyl)-3-methyl-2,4-pentadienoic acid was metabolized by Cercospora cruenta, which has the ability to produce (+)-abscisic acid (ABA), to give (±)-(2Z,4E)-xanthoxin acid, (±)-(2Z,4E)-5′-hydroxy-1′,2′-epoxy-1′,2′-dihydro-β-ionylideneacetic acid, (±)-1′,2′-epoxy-1′,2′-dihydro-β-ionone and trace amounts of ABA.     

Identification of a plausible biosynthetic enzyme for the IM-2-type autoregulator in Streptomyces antibioticus

Virginiae butanolides (VBs) and IM-2 are members of Streptomyces hormones called ‘butyrolactone autoregulators’ which regulate the antibiotic production in Streptomyces species at nanomolar concentrations. Cell-free extract of a VB-A overproducer, Streptomyces antibioticus NF-18, is capable of catalyzing the final step of the autoregulator biosynthesis, namely, the NADPH-dependent reduction of 6-dehydroVB-A. However, physico-chemical analyses of the purified enzymatic products revealed that, in addition to the VB-type isomer [(2R,3R,6S)-enantiomer], IM-2-type isomers [(2R,3R,6R)- and (2S,3S,6S)-enantiomers] were also produced from (±)-6-dehydroVB-A, suggesting the existence of several 6-dehydroVB-A reductases with respective stereoselectivities. The reductase activity of the crude extracts was separated into two activity peaks, peak I (major) and peak II (minor), by DEAE-5PW HPLC. Chiral HPLC analyses demonstrated that peak I enzyme and peak II enzyme catalyzed the production of (2R,3R,6S), (2R,3R,6R) and (2S,3S,6S) isomers at ratios of 46:1:3.2 and 4.9:1:1.5, respectively, indicating clearly that S. antibioticus NF-18 possesses at least two 6-dehydroVB-A reductases: one much favored toward VB-A biosynthesis, the other with relaxed stereoselectivity capable of synthesizing both VB-type and IM-2-type autoregulators.     

Characterization of an NAD(P)+-dependent meso-diaminopimelate dehydrogenase from Thermosyntropha lipolytica

meso-Diaminopimelate dehydrogenase (meso-DAPDH) catalyzes the reversible NADP⁺-dependent oxidative deamination of meso-2,6-diaminopimelate (meso-DAP) to produce l-2-amino-6-oxopimelate. meso-DAPDH is divided into two major clusters, types I and II, based on substrate specificity and structural characteristic. Here, we describe a novel type II meso-DAPDH from Thermosyntropha lipolytica (TlDAPDH). The gene encoding a putative TlDAPDH was expressed in Escherichia coli cells, and then the enzyme was purified 7.3-fold to homogeneity from the crude cell extract. The molecule of TlDAPDH seemed to form a hexamer, which is the typical structural characteristic of type II meso-DAPDHs. The purified enzyme exhibited oxidative deamination activity toward meso-DAP with both NADP⁺ and NAD⁺ as coenzymes. TlDAPDH exhibited reductive amination activity of corresponding 2-oxo acid to produce d-amino acid. In particular, the productivities for d-aspartate and d-glutamate have not been reported in the type II enzymes. The optimum pH and temperature for oxidative deamination of meso-DAP were 10.5 and 55°C, respectively. TlDAPDH retained more than 80% of its activity after incubation for 30 min at temperatures between 50°C and 65°C and in the pH range of 4.5–9.5. Moreover, the coenzyme and substrate recognition mechanisms of TlDAPDH were elucidated based on a multiple sequence alignment and the homology model. The results of these analyses suggested that the molecular mechanisms for coenzyme and substrate recognition of TlDAPDH were similar to those of meso-DAPDH from S. thermophilum, which is the representative type II enzyme. Based on the kinetic characteristics and structural comparison, TlDAPDH was considered to be a novel type II meso-DAPDH.     

The allantoinase of Lathyrus sativus

The presence of a stable allantoinase in Lathyrus sativas and its de novo synthesis at a maximal rate in the first 48 hr of germination have been demonstrated. The plumule and radicle together exhibited highest enzyme activity. L. sativas allantoinase has been purified nearly 35-fold. The purified enzyme was optically active around pH 7.5, did not require any metal ion for activity and exhibited a K_m of 2·56 mM for (±)-allantoin, and an activation energy of 5·6 kcal/mol. Unlike other plant allantoinases, the L. sativus enzyme is highly specific for (±)-allantoin and is shown to be a sulfhydryl enzyme which apparently exists in a stable form in vivo obviating the need for added sulfhydryl compounds for maximal activity.     

Promotive effect of chrysanthemic acids on adventitious root formation in some plants

Adventitious root formation in excised cucumber (Cucumis sativus L.) cotyledons was significantly promoted by (±)-cis-chrysanthemic acid at 0.006–1.8 mM. The effect of (±)-cis-chrysanthemic acid on indole-3-acetic acid (IAA)-induced rooting was additive. Rooting in excised cucumber cotyledons was significantly promoted by several isomers of chrysanthemic acid and sodium (±)-cis-chrysanthemate at 0.18 mM. Rooting in mung bean (Phaseolus radiatus L.) hypocotyls was also stimulated by the sodium salt at 0.06–0.6 mM. Rooting of kidney bean (Phaseolus vulgaris L.) hypocotyls was also clearly enhanced by sodium (±)-cis-chrysanthemate at 0.18–6 mM.     

Syntheses of benzophenone-xanthone hybrid polyketides and their antibacterial activities

Muchimangins are benzophenone-xanthone hybrid polyketides produced by Securidaca longepedunculata. However, their biological activities have not been fully investigated, since they are minor constituents in this plant. To evaluate the possibility of muchimangins as antibacterial agent candidates, five muchimangin analogs were synthesized from 2,4,5-trimethoxydiphenyl methanol and the corresponding xanthones, by utilizing p-toluenesulfonic acid monohydrate for the Brønsted acid-catalysis. The antibacterial assays against Gram-positive bacteria, Staphylococcus aureus and Bacillus subtilis, and Gram-negative bacteria, Klebsiella pneumoniae and Escherichia coli, revealed that the muchimangin analogs (±)-1,3,6,8-tetrahydroxy-4-(phenyl-(2′,4′,5′-trimethoxyphenyl)methyl)-xanthone (1), (±)-1,3,6-trihydroxy-4-(phenyl-(2′,4′,5′-trimethoxyphenyl)methyl)-xanthone (2), and (±)-1,3-dihydroxy-4-(phenyl-(2′,4′,5′-trimethoxyphenyl)methyl)-xanthone (3) showed significant activities against S. aureus, with MIC values of 10.0, 10.0, and 25.0 μM, respectively. Analogs (±)-1 and (±)-2 also exhibited antibacterial activities against B. subtilis, with MIC values of 50.0 and 12.5 μM, respectively. Furthermore, (+)-3 enhanced the antibacterial activity against S. aureus, with a MIC value of 10 μM.     

Synthesis,in-vitro α-glucosidase inhibition,antioxidant, in-vivo antidiabetic and molecular docking studies of pyrrolidine-2,5-dione and thiazolidine-2,4-dione derivatives

α-Glucosidase is considered as a therapeutic target for the treatment of type 2 diabetes mellitus (DM2). In current study, we synthesized pyrrolidine-2,5-dione (succinimide) and thiazolidine-2,4-dione derivatives and evaluated for their ability to inhibit α-Glucosidase. Pyrrolidine-2,5-dione derivatives (11a–o) showed moderate to poor α-glucosidase inhibition. Compound 11o with the IC₅₀ value of 28.3 ± 0.28 µM emerged as a good inhibitor of α-glucosidase. Thiazolidine-2,4-dione and dihydropyrimidine (TZD-DHPM) hybrids (22a–c) showed excellent inhibitory activities. The most active compound 22a displayed IC₅₀ value of 0.98 ± 0.008 µM. Other two compounds of this series also showed activity in low micromolar range. The in-vivo antidiabetic study of three compounds 11n, 11o and 22a were also determined using alloxan induced diabetes mice model. Compounds 11o and 22a showed significant hypoglycemic effect compared to the reference drug. In-vivo acute toxicity study showed the safety of these selected compounds. In-silico docking studies were carried out to rationalize the in-vitro results. The binding modes and bioassay results of TZD-DHPM hybrids showed that interactions with important residues appeared significant for high potency.     

Pterocarpans from Dalbergia spruceana

The wood of Dalbergia spruceana contains, besides O-acetyloleanolic acid, elemicin and 3,4,5-trimethoxy-cinnamaldehyde; the neoflavonoids (S)-4-methoxydalbergione and dalbergin; the isoflavones biochanin-A, formononetin, pseudobaptigenin and caviunin; and the pterocarpans (±)-medicarpin, (±)-maackiain, as well as the 3,4-dihydroxy-, 3-hydroxy-4-methoxy- and 4-hydroxy-3-methoxy-derivatives of (6aR,11aR)-8,9-methylenedioxypterocarpan. The constitutions of the three last named compounds were deduced by spectra and confirmed by synthesis of (±)-4-hydroxy-3-methoxy-8,9-methylenedioxypterocarpan.     

Biosynthesis of the bisbenzylisoquinoline alkaloid,tetrandrine

The incorporation of (±)-coclaurine, (±)-norcoclaurine, (±)-N-methylcoclaurine and didehydro-N-methyleoclaurinium iodide into tetrandrine in Cocculus laurifolius has been studied and specific utilization of (±)-N-ethylcoclaurine demonstrated. The evidence indicates that tetrandrine is formed in the plants by oxidative dimerization of N-methylcoclaurine. Double labelling experiment with (±)-N- [¹⁴C]-methyl- [1-³H]-coclaurine demonstrated that the hydrogen atom at the asymmetric centre in the 1-benzylisoquinoline precursor is retained in the bioconversion into tetrandrine. Parallel feedings of (+)-(S)- and (?)-(R)-N-methylcoclaurines showed that the stereospecificity is maintained in the biosynthesis of tetrandrine from the 1-benzylisoquinoline precursor.     

Isomerization and Racemization of Menthols

The hitherto unknown cyanoacetates of menthol isomers, i.e., (±)-menthyl, (±)- and (+)-neomenthyl, and (±)- and (+)-isomenthyl cyanoacetate, have been prepared. It has been proved to be possible to isolate effectively pure (±)-menthol from optically inactive mixture of isomers through the cyanoacetate, followed by saponification.