Determination of plasma and serum l-lysine using l-lysine ε-oxidase from Marinomonas mediterranea NBRC 103028

This article describes a successful application of l-lysine ε-oxidase (EC 1.4.3.20) for l-lysine determination. l-Lysine ε-oxidase was isolated from culture supernatant of Marinomonas mediterranea NBRC 103028^T and was used for l-lysine determination. Comparison of the characteristics of l-lysine ε-oxidase with l-lysine α-oxidase, a commercial enzyme used for l-lysine determination, suggests that the use of l-lysine ε-oxidase would be more valuable for the determination of l-lysine because of its selectivity and sensitivity, especially in samples with low l-lysine concentration. The enzyme acted only on l-lysine and l-ornithine, to which the relative activity was only 3.4% of that on l-lysine. The value obtained by the colorimetric assay using l-lysine ε-oxidase and horseradish peroxidase was not affected by l-ornithine. The enzyme also shows a higher affinity for l-lysine (K_m = 0.0018 mM). l-Lysine determination using l-lysine ε-oxidase in human plasma and serum was examined. The measured values were close to values determined by instrumental analyses using the precolumn AccQ·Tag Ultra Derivatization Kit. These results suggest that l-lysine ε-oxidase can be used for diagnosis based on plasma l-lysine concentration. This is the first report on the application of l-lysine ε-oxidase.     

Antibacterial activity and mechanism of action of ε-poly-l-lysine

ε-Poly-l-lysine (ε-PL)² is widely used as an antibacterial agent because of its broad antimicrobial spectrum. However, the mechanism of ε-PL against pathogens at the molecular level has not been elucidated. This study investigated the antibacterial activity and mechanism of ε-PL against Escherichia coli O157:H7 CMCC44828. Propidium monoazide-PCR test results indicated that the threshold condition of ε-PL for complete membrane lysis of E. coli O157:H7 was 10 μg/mL (90% mortality for 5 μg/mL). Further verification of the destructive effect of ε-PL on cell structure was performed by atomic force microscopy and transmission electron microscopy. Results showed a positive correlation between reactive oxygen species (ROS) ³ levels and ε-PL concentration in E. coli O157:H7 cells. Moreover, the mortality of E. coli O157:H7 was reduced when antioxidant N-acetylcysteine was added. Results from real-time quantitative PCR (RT-qPCR) ⁴ indicated that the expression levels of oxidative stress genes sodA and oxyR were up-regulated 4- and 16-fold, respectively, whereas virulence genes eaeA and espA were down-regulated after ε-PL treatment. Expression of DNA damage response (SOS response) ⁵ regulon genes recA and lexA were also affected by ε-PL. In conclusion, the antibacterial mechanism of ε-PL against E. coli O157:H7 may be attributed to disturbance on membrane integrity, oxidative stress by ROS, and effects on various gene expressions, such as regulation of oxidative stress, SOS response, and changes in virulence.     

The synthesis of a mannosyl-N-acetylglucosamine-l-asparagine compound: 2-acetamido-N-(l-aspart-4-oyl)-2-deoxy-3-O-α-d-mannopyranosyl-β-d-glucopyranosylamine

The title compound, used in the synthesis of glycopeptides and as a reference substance in the structural elucidation of glycoproteins, was synthesized by condensation of 2,3,4,6-tetra-O-acetyl-α-d-mannopyranosyl bromide with 2-acetamido-4,6-O-benzylidene-α-d-glucopyranosyl azide, followed by removal of the benzylidene group to give the disaccharide azide 6 and acetylation. The resulting fully acetylated disaccharide azide 7 was also obtained by treatment of the known 2-acetamido-1,4,6-tri-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-α-d-mannopyranosyl)-α-d-glucopyranose with hydrogen chloride and then with silver azide. The azide 7 was reduced in presence of platinum oxide (Adams' catalyst), and the resulting amine was condensed with 1-benzyl N-benzyloxycarbonyl-l-aspartate in the presence of N,N′-dicyclocarbodiimide. The removal of the protective group was accomplished by hydrogenolysis and O-deacetylation. In a second route, the disaccharide azide 6 was reduced and then condensed with 1-benzyl N-benzyloxycarbonyl-l-aspartate, and the resulting product hydrogenolyzed.     

Physicochemical and molecular modeling studies of cefixime–l-arginine–cyclodextrin ternary inclusion compounds

In an attempt to improve the physicochemical properties of cefixime (CEF), its supramolecular inclusion compounds were prepared with β-cyclodextrin (βCD) and hydroxypropyl-β-cyclodextrin (HPβCD) in presence and/or absence of ternary component l-arginine (ARG) using spray drying technique. Initially, the phase solubility studies revealed a stoichiometry of 1:1 molar ratio with an A_L-type of phase solubility curve. The stability constants of binary systems were remarkably improved in presence of ARG, indicating positive effect of its addition. The inclusion complexes were characterized by FTIR, XRPD, DSC, SEM, particle size analysis, and dissolution studies. Further, molecular mechanic (MM) calculations were performed to investigate the possible orientations of CEF inside βCD cavity in presence and/or absence of ternary component. In case of physicochemical studies, the ternary systems performed well as a result of comprehensive effect of ternary complexation and particle size reduction achieved by a spray drying technology.     

A novel C–S lyase from the latex-producing plant Taraxacum brevicorniculatum displays alanine aminotransferase and l-cystine lyase activity

We isolated a novel pyridoxal-5-phosphate-dependent l-cystine lyase from the dandelion Taraxacum brevicorniculatum. Real time qPCR analysis showed that C–S lyase from Taraxacum brevicorniculatum (TbCSL) mRNA is expressed in all plant tissues, although at relatively low levels in the latex and pedicel. The 1251 bp TbCSL cDNA encodes a protein with a calculated molecular mass of 46,127 kDa. It is homologous to tyrosine and alanine aminotransferases (AlaATs) as well as to an Arabidopsis thaliana carbon–sulfur lyase (C–S lyase) (SUR1), which has a role in glucosinolate metabolism. TbCSL displayed in vitrol-cystine lyase and AlaAT activities of 4 and 19 nkat mg⁻¹ protein, respectively. However, we detected no in vitro tyrosine aminotransferase (TyrAT) activity and RNAi knockdown of the enzyme had no effect on phenotype, showing that TbCSL substrates might be channeled into redundant pathways. TbCSL is in vivo localized in the cytosol and functions as a C–S lyase or an aminotransferase in planta, but the purified enzyme converts at least two substrates specifically, and can thus be utilized for further in vitro applications.     

Synthesis of dopamine and l-DOPA-α-glycosides by reaction with cyclomaltohexaose catalyzed by cyclomaltodextrin glucanyltransferase

Dopamine-HCl and l-DOPA-α-glycosides were prepared by reaction with cyclomaltohexaose, catalyzed by Bacillus macerans cyclomaltodextrin glucanyltransferase. The reaction gave maltodextrins attached to dopamine and l-DOPA; the maltodextrins were trimmed by reactions with glucoamylase and β-amylase to produce α-glucosyl- and α-maltosyl-glycosides, respectively. The glucoamylase- or β-amylase-treated dopamine- and l-DOPA-α-glycosides were fractionated and purified by BioGel P-2 gel-filtration column chromatography and preparative descending paper chromatography. Analysis by MALDI-TOF mass spectrometry and one- and two-dimensional NMR showed that the purified glycosides of dopamine and l-DOPA were glycosylated at the hydroxyl groups of positions 3 and 4 of the catechol ring. The major product was found to be 4-O-α-glycopyranosyl l-DOPA, and it was shown to be more resistant to oxidative tolerance experiments, involving hydrogen peroxide and ferrous ion, than l-DOPA. l-DOPA-α-glycosides are possibly more effective substitutes for l-DOPA in treating Parkinson’s disease in that they are more resistant to oxidation and methylation, which renders l-DOPA ineffective and deleterious.     

Crystal structure of glycoside hydrolase family 127 β-l-arabinofuranosidase from Bifidobacterium longum

Enzymes acting on β-linked arabinofuranosides have been unknown until recently, in spite of wide distribution of β-l-arabinofuranosyl oligosaccharides in plant cells. Recently, a β-l-arabinofuranosidase from the glycoside hydrolase family 127 (HypBA1) was discovered in the newly characterized degradation system of hydroxyproline-linked β-l-arabinooligosaccharides in the bacterium Bifidobacterium longum. Here, we report the crystal structure of HypBA1 in the ligand-free and β-l-arabinofuranose complex forms. The structure of HypBA1 consists of a catalytic barrel domain and two additional β-sandwich domains, with one β-sandwich domain involved in the formation of a dimer. Interestingly, there is an unprecedented metal-binding motif with Zn²⁺ coordinated by glutamate and three cysteines in the active site. The glutamate residue is located far from the anomeric carbon of the β-l-arabinofuranose ligand, but one cysteine residue is appropriately located for nucleophilic attack for glycosidic bond cleavage. The residues around the active site are highly conserved among GH127 members. Based on biochemical experiments and quantum mechanical calculations, a possible reaction mechanism involving cysteine as the nucleophile is proposed.     

Selective purification of supercoiled p53-encoding pDNA with l-methionine–agarose matrix

The p53 tumor suppressor gene has been widely explored for gene therapy as an alternative to the common treatments. Recently, the supercoiled conformation of a p53-encoding plasmid proved to be more efficient in cell transfection and protein expression than the open circular conformation. To successfully isolate this isoform, several chromatographic techniques have been used, namely affinity chromatography with amino acids as ligands. However, the study of new matrices and ligands with higher specificity and robustness for supercoiled plasmid purification is still required. The present work explores for the first time a new matrix of l-methionine–agarose to efficiently purify the supercoiled p53-encoding plasmid. The binding/elution conditions, such as salt concentration and temperature, were manipulated and combined to attain the best strategy. Therefore, the supercoiled plasmid isoform was purified from a clarified lysate by using a decreasing stepwise gradient comprising 2.35 and 1.7 M ammonium sulfate in 10 mM Tris–HCl, pH 8.0, and finally 10 mM Tris–HCl, pH 8.0, at 5 °C. After accomplishing the purification process, we performed several tests to assess the quality of the supercoiled plasmid, revealing that the amounts of proteins, gDNA, RNA, and endotoxins were significantly reduced or undetectable in the final formulation.     

β-Xylosidases and α-l-arabinofuranosidases: Accessory enzymes for arabinoxylan degradation

Arabinoxylan (AX) is among the most abundant hemicelluloses on earth and one of the major components of feedstocks that are currently investigated as a source for advanced biofuels. As global research into these sustainable biofuels is increasing, scientific knowledge about the enzymatic breakdown of AX advanced significantly over the last decade. This review focuses on the exo-acting AX hydrolases, such as α-arabinofuranosidases and β-xylosidases. It aims to provide a comprehensive overview of the diverse substrate specificities and corresponding structural features found in the different glycoside hydrolase families. A careful review of the available literature reveals a marked difference in activity between synthetically labeled and naturally occurring substrates, often leading to erroneous enzymatic annotations. Therefore, special attention is given to enzymes with experimental evidence on the hydrolysis of natural polymers.     

Functional assignment of Glu386 and Arg388 in the active site of l-galactono-γ-lactone dehydrogenase

The flavoenzyme l-galactono-γ-lactone dehydrogenase (GALDH) catalyzes the terminal step of vitamin C biosynthesis in plants. Little is known about the catalytic mechanism of GALDH and related aldonolactone oxidoreductases. Here we identified an essential Glu-Arg pair in the active site of GALDH from Arabidopsis thaliana. Glu386 and Arg388 variants show high K_m values for l-galactono-1,4-lactone and low turnover rates. Arg388 is crucial for the stabilization of the anionic form of the reduced FAD cofactor. Glu386 is involved in productive substrate binding. The E386D variant has lost its specificity for l-galactono-1,4-lactone and shows the highest catalytic efficiency with l-gulono-1,4-lactone.     

Analysis of l-serine-O-phosphate in cerebrospinal spinal fluid by derivatization–liquid chromatography/mass spectrometry

l-Serine-O-phosphate (l-SOP), the precursor of l-serine, is a potent agonist against the group III metabotropic glutamate receptors (mGluRs) and, thus, is of interest as a potential biomarker for monitoring modulation of neurotransmitter release. So far, no reports are available on the analysis of l-SOP in cerebrospinal fluid (CSF). Here a novel method is presented to determine l-SOP levels in CSF employing precolumn derivatization with (5-N-succinimidoxy-5-oxopentyl)triphenylphosphonium bromide (SPTPP) coupled to liquid chromatography/mass spectrometry (derivatization–LC/MS, d-LC/MS).     

X-ray structure of 1,3,4-tri-O-acetyl-2-deoxy-β-d-erythro-pentopyranose

Peracetylated 2-deoxy-d-erythro-pentose (2-deoxy-d-ribose) was synthesized through the acetylation of 2-deoxy-d-ribose with acetic anhydride in pyridine, and the products (including all four ring forms) exist in form of either a white solid or a syrup. A single crystal of 1,3,4-tri-O-acetyl-2-deoxy-β-d-erythro-pentopyranose was obtained from the syrup and its structure was determined by X-ray diffraction. The crystal adopts the ¹C₄ conformation, presenting an orthorhombic system, space group P2₁2₁2₁ with Z = 4, unit cell dimensions a = 7.2274 (3) Å, b = 8.0938 (5) Å, and c = 22.0517 (11) Å.     

Targeted delivery of paclitaxel using folate-conjugated heparin-poly(β-benzyl-l-aspartate) self-assembled nanoparticles

A self-assembled nanoparticulate system composed of a folate-conjugated heparin-poly(β-benzyl-l-aspartate) (HP) amphiphilic copolymer was proposed for targeted delivery of the antineoplastic drug paclitaxel (PTX). PTX was incorporated into three types of heparin-based nanoparticles, including HP, folate-conjugated HP (FHP), and folate-polyethylene glycol (PEG)-conjugated HP (FPHP), using a simple dialysis method. The PTX-loaded nanoparticles were then characterized according to particle size (140-190 nm) and size distribution, drug-loading content and efficiency, and in vitro release behavior. In the cellular uptake study using KB cells positive for the folate-receptor (FR), FHP and FPHP nanoparticles showed a much higher cellular uptake than did unconjugated HP nanoparticles. Specifically, when the PEG spacer was inserted between the folate ligand and heparin backbone, FPHP nanoparticles had a greater cellular uptake than did FHP nanoparticles. The in vitro cytotoxicity of PTX-loaded HP, FHP, and FPHP nanoparticles was studied in KB cells and FR-negative A549 cells. Compared with the cytotoxicity in A549 cells, PTX-loaded FHP and FPHP nanoparticles exhibited more potent cytotoxicity in KB cells than did PTX-loaded HP nanoparticles and free-PTX, suggesting that the presence of folate enhanced intracellular uptake via FR-mediated endocytosis. In addition, FPHP nanoparticles exhibited much greater cytotoxicity in KB cells than did FHP nanoparticles. These results suggest that PTX-loaded folate-conjugated HP nanoparticles are a potentially useful delivery system for cancer cells positive for the folate-receptor.     

Monoacylation of 2-O-α-d-glucopyranosyl-l-ascorbic acid by protease in N,N-dimethylformamide with low water content

2-O-α-d-Glucopyranosyl-l-ascorbic acid (AA-2G) laurate was synthesized from AA-2G and vinyl laurate with a protease from Bacillus subtilis in N,N-dimethylformamide (DMF) with low water content. Addition of water to DMF dramatically enhanced monoacyl AA-2G synthesis. Maximum synthetic activity was observed when 3% (v/v) water was added to the reaction medium. Under the optimal reaction conditions, 5-O-dodecanoyl-2-O-α-d-glucopyranosyl-l-ascorbic acid, 2-O-(6′-O-dodecanoyl-α-d-glucopyranosyl)-l-ascorbic acid, and 6-O-dodecanoyl-2-O-α-d-glucopyranosyl-l-ascorbic acid were synthesized in yields of 5.5%, 3.2%, and 20.4%, respectively.     

Nucleophilic displacement reactions in carbohydrates : Part XX. Displacements on alkyl α-l-talopyranoside 4-sulphonate derivatives

The azide displacement reaction on methyl 6-deoxy-4-O-methanesulphonyl-2,3-di-O-methyl-α-l-talopyranoside (6) in N,N-dimethylformamide yielded methyl 4,6-dideoxy-2,3-di-O-methyl-α-l-threo-hex-3-enopyranoside (7, ca. 50%), methyl 4,6-dideoxy-2,3-di-O-methyl-β-d-erythro-hex-4-enopyranoside (8, ca. 10%), and methyl 4-azido-4,6-dideoxy-2,3-di-O-methyl-α-l-mannopyranoside (9, ca. 40%). The corresponding azide 14 (20%) and the unsaturated sugars 12 (68%) and 13 (12%) were obtained from a comparable reaction on benzyl 6-deoxy-4-O-methanesulphonyl-2,3-di-O-methyl-α-l-talopyranoside (11).     

Dimeric Crystal Structure of Rabbit l-Gulonate 3-Dehydrogenase/λ-Crystallin: Insights into the Catalytic Mechanism

l-Gulonate 3-dehydrogenase (GDH) is a bifunctional dimeric protein that functions not only as an NAD⁺-dependent enzyme in the uronate cycle but also as a taxon-specific λ-crystallin in rabbit lens. Here we report the first crystal structure of GDH in both apo form and NADH-bound holo form. The GDH protomer consists of two structural domains: the N-terminal domain with a Rossmann fold and the C-terminal domain with a novel helical fold. In the N-terminal domain of the NADH-bound structure, we identified 11 coenzyme-binding residues and found 2 distinct side-chain conformers of Ser124, which is a putative coenzyme/substrate-binding residue. A structural comparison between apo form and holo form and a mutagenesis study with E97Q mutant suggest an induced-fit mechanism upon coenzyme binding; coenzyme binding induces a conformational change in the coenzyme-binding residues Glu97 and Ser124 to switch their activation state from resting to active, which is required for the subsequent substrate recruitment. Subunit dimerization is mediated by numerous intersubunit interactions, including 22 hydrogen bonds and 104 residue pairs of van der Waals interactions, of which those between two cognate C-terminal domains are predominant. From a structure/sequence comparison within GDH homologues, a much greater degree of interprotomer interactions (both polar and hydrophobic) in the rabbit GDH would contribute to its higher thermostability, which may be relevant to the other function of this enzyme as λ-crystallin, a constitutive structural protein in rabbit lens. The present crystal structures and amino acid mutagenesis studies assigned the role of active-site residues: catalytic base for His145 and substrate binding for Ser124, Cys125, Asn196, and Arg231. Notably, Arg231 participates in substrate binding from the other subunit of the GDH dimer, indicating the functional significance of the dimeric state. Proper orientation of the substrate-binding residues for catalysis is likely to be maintained by an interprotomer hydrogen-bonding network of residues Asn196, Gln199, and Arg231, suggesting a network-based substrate recognition of GDH.     

Synthesis and conformational analysis of carbasugar bioisosteres of α-l-iduronic acid and its methyl glycoside

The synthesis of two novel carbasugar analogues of α-l-iduronic acid is described in which the ring-oxygen is replaced by a methylene group. In analogy with the conformational equilibrium described for α-l-IdopA, the conformation of the carbasugars was investigated by ¹H and ¹³C NMR spectroscopy. Hadamard transform NMR experiments were utilised for rapid acquisition of ¹H,¹³C-HSQC spectra and efficient measurements of heteronuclear long-range coupling constants. Analysis of ¹H NMR chemical shifts and J_H,H coupling constants extracted by a total-lineshape fitting procedure in conjunction with J_H,C coupling constants obtained by three different 2D NMR experiments, viz., ¹H,¹³C-HSQC-HECADE, J-HMBC and IPAP-HSQC-TOCSY-HT, as well as effective proton-proton distances from 1D ¹H,¹H T-ROE and NOE experiments showed that the conformational equilibrium ⁴C₁?²S_5a?¹C₄ is shifted towards ⁴C₁ as the predominant or exclusive conformation. These carbasugar bioisosteres of α-l-iduronic acid do not as monomers show the inherent flexibility that is anticipated to be necessary for biological activity.     

Enzymic synthesis of glutamic acid γ-semialdehyde (Δ1-pyrroline-5-carboxylate) and N-acetyl-l-glutamic acid γ-semialdehyde: Isolation and characterization of their 2,4-dinitrophenylhydrazones

A protocol is given for the synthesis, by a simple enzyme preparation, of the l-isomers of glutamic acid γ-semialdehyde and N-acetylglutamic acid γ-semialdehyde. The isolation and physical and chemical properties of these products as the 2,4-dinitrophenylhydrazone derivatives are described. Spectral characteristics, NMR, ir, and visible, and melting points for these compounds are given.