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.     

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.     

Crystal Structure and Site-directed Mutagenesis of 3-Ketosteroid Δ1-Dehydrogenase from Rhodococcus erythropolis SQ1 Explain Its Catalytic Mechanism

3-Ketosteroid Δ¹-dehydrogenases are FAD-dependent enzymes that catalyze the 1,2-desaturation of 3-ketosteroid substrates to initiate degradation of the steroid nucleus. Here we report the 2.0 Å resolution crystal structure of the 56-kDa enzyme from Rhodococcus erythropolis SQ1 (Δ¹-KSTD1). The enzyme contains two domains: an FAD-binding domain and a catalytic domain, between which the active site is situated as evidenced by the 2.3 Å resolution structure of Δ¹-KSTD1 in complex with the reaction product 1,4-androstadiene-3,17-dione. The active site contains four key residues: Tyr¹¹⁹, Tyr³¹⁸, Tyr⁴⁸⁷, and Gly⁴⁹¹. Modeling of the substrate 4-androstene-3,17-dione at the position of the product revealed its interactions with these residues and the FAD. The C1 and C2 atoms of the substrate are at reaction distance to the N5 atom of the isoalloxazine ring of FAD and the hydroxyl group of Tyr³¹⁸, respectively, whereas the C3 carbonyl group is at hydrogen bonding distance from the hydroxyl group of Tyr⁴⁸⁷ and the backbone amide of Gly⁴⁹¹. Site-directed mutagenesis of the tyrosines to phenylalanines confirmed their importance for catalysis. The structural features and the kinetic properties of the mutants suggest a catalytic mechanism in which Tyr⁴⁸⁷ and Gly⁴⁹¹ work in tandem to promote keto-enol tautomerization and increase the acidity of the C2 hydrogen atoms of the substrate. With assistance of Tyr¹¹⁹, the general base Tyr³¹⁸ abstracts the axial β-hydrogen from C2 as a proton, whereas the FAD accepts the axial α-hydrogen from the C1 atom of the substrate as a hydride ion.     

Crystal Structures of Human CaMKIα Reveal Insights into the Regulation Mechanism of CaMKI

Human calcium/calmodulin-dependent protein kinase I (CaMKI) plays pivotal roles in the nervous system. The activity of human CaMKI is regulated by a regulatory region including an autoinhibitory segment and a CaM-binding segment. We report here four structures of three CaMKIα truncates in apo form and in complexes with ATP. In an apo, autoinhibited structure, the activation segment adopts a unique helical conformation which together with the autoinhibitory segment constrains helices αC and αD in inactive conformations, sequesters Thr177 from being phosphorylated, and occludes the substrate-binding site. In an ATP-bound, inactive structure, the activation segment is largely disordered and the CaM-binding segment protrudes out ready for CaM binding. In an ATP-bound, active structure, the regulatory region is dissociated from the catalytic core and the catalytic site assumes an active conformation. Detailed structural analyses reveal the interplay of the regulatory region, the activation segment, and the nucleotide-binding site in the regulation of CaMKI.     

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.     

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.     

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.     

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.     

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.     

Formation and structure in aqueous solution of complexes between vanadium(V) and aminohydroxamic acids that potentiates vanadium’s insulinomimetic activity: l-glutamic γ-hydroxamic and l-aspartic-β-hydroxamic acids

The full speciation of the vanadium(V) complexation systems with two aminohydroxamic acids, aspartic-β- and glutamic-γ-hydroxamic acid, has been determined using potentiometric and spectroscopic techniques. Formation constants were calculated in a systematic study at different ligand to metal molar ratios and the coordination types are proposed. An almost constant value of the ⁵¹V NMR signal in neutral medium can be attributed to two (1:1 and 1:2 metal to ligand ratios) similar structures, both of which can be either protonated or deprotonated. The two ligands have a carboxylic group in the structure and show comparable biological activities. In this work analogous complexation behavior at physiological conditions was found despite the presence of two or three methylenic groups between the amino and hydroxamate groups. The carboxylic groups are quite distant from the hydroxamic groups and are not involved directly in the coordination process. Therefore the coordination structures are related to that found in the vanadium(V)-β-alaninehydroxamic acid in which there is not a carboxylic group.     

Novel Insights into Eukaryotic γ-Glutamyltranspeptidase 1 from the Crystal Structure of the Glutamate-bound Human Enzyme

The enzyme γ-glutamyltranspeptidase 1 (GGT1) is a conserved member of the N-terminal nucleophile hydrolase family that cleaves the γ-glutamyl bond of glutathione and other γ-glutamyl compounds. In animals, GGT1 is expressed on the surface of the cell and has critical roles in maintaining cysteine levels in the body and regulating intracellular redox status. Expression of GGT1 has been implicated as a potentiator of asthma, cardiovascular disease, and cancer. The rational design of effective inhibitors of human GGT1 (hGGT1) has been delayed by the lack of a reliable structural model. The available crystal structures of several bacterial GGTs have been of limited use due to differences in the catalytic behavior of bacterial and mammalian GGTs. We report the high resolution (1.67 Å) crystal structure of glutamate-bound hGGT1, the first of any eukaryotic GGT. Comparisons of the active site architecture of hGGT1 with those of its bacterial orthologs highlight key differences in the residues responsible for substrate binding, including a bimodal switch in the orientation of the catalytic nucleophile (Thr-381) that is unique to the human enzyme. Compared with several bacterial counterparts, the lid loop in the crystal structure of hGGT1 adopts an open conformation that allows greater access to the active site. The hGGT1 structure also revealed tightly bound chlorides near the catalytic residue that may contribute to catalytic activity. These are absent in the bacterial GGTs. These differences between bacterial and mammalian GGTs and the new structural data will accelerate the development of new therapies for GGT1-dependent diseases.     

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).     

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.     

Presence of 1→3-linked 2-O-β-d-xylopyranosyl-α-l-arabinofuranosyl side chains in cereal arabinoxylans

The presence of a fairly uncommon side chain 2-O-β-d-xylopyranosyl-α-l-arabinofuranosyl in arabinoxylans (AX) from eight different cereal by-products was investigated, using ¹H NMR spectroscopy and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) after Shearzyme^® (GH10 endo-1,4-β-d-xylanase) hydrolysis. This disaccharide side group was present in significant amounts in AX extracted from corn cobs and barley husks. For the first time, it was also detected in AX from oat spelts and rice husks, and in lesser amounts in wheat straw AX. Arabinoxylo-oligosaccharide (AXOS) containing the 2-O-β-d-Xylp-α-l-Araf side chain was purified from the oat spelt AX hydrolysate and the structure was fully analyzed using 1D and 2D NMR spectroscopy. The AXOS was identified as β-d-Xylp-(1→2)-α-l-Araf-(1→3)-β-d-Xylp-(1→4)-d-Xyl. To our knowledge, such a structure with 2-O-β-d-Xylp-α-l-Araf attached to the O-3 of the nonreducing end of xylobiose has not been described previously. New information on substitution of AX from various cereal by-products was obtained by combining NMR and enzyme-assisted HPAEC-PAD analysis.     

Short synthetic sequence for 2-sulfation of α-l-iduronate glycosides

Hunter syndrome (mucopolysaccharidosis-II) is caused by deficiency of the lysosomal enzyme iduronate-2-sulfatase. The assay of this sulfatase requires the use of α-l-iduronate glycosides containing a sulfate at the 2-position. We report a simple, three-step procedure for the introduction of sulfate at the 2-position starting with the methyl ester of α-l-iduronate glycosides. The procedure involves protection of the 2- and 4-hydroxyl groups of the iduronate moiety as the dibutyl stannylene acetal, selective sulfation with sulfur trioxide-trimethylamine, and deprotection of the methyl ester to afford the desired 2-sulfate in 61% overall yield.     

A H NMR study of the specificity of α-l-arabinofuranosidases on natural and unnatural substrates

Background

The detailed characterization of arabinoxylan-active enzymes, such as double-substituted xylan arabinofuranosidase activity, is still a challenging topic. Ad hoc chromogenic substrates are useful tools and can reveal subtle differences in enzymatic behavior. In this study, enzyme selectivity on natural substrates has been compared with enzyme selectivity towards aryl-glycosides. This has proven to be a suitable approach to understand how artificial substrates can be used to characterize arabinoxylan-active α-l-arabinofuranosidases (Abfs).

Methods

Real-time NMR using a range of artificial chromogenic, synthetic pseudo-natural and natural substrates was employed to determine the hydrolytic abilities and specificity of different Abfs.

Results

The way in which synthetic di-arabinofuranosylated substrates are hydrolyzed by Abfs mirrors the behavior of enzymes on natural arabinoxylo-oligosaccharide (AXOS). Family GH43 Abfs that are strictly specific for mono-substituted d-xylosyl moieties (AXH-m) do not hydrolyze synthetic di-arabinofuranosylated substrates, while those specific for di-substituted moieties (AXH-d) remove a single l-arabinofuranosyl (l-Araf) group. GH51 Abfs, which are supposedly AXH-m enzymes, can release l-Araf from disubstituted d-xylosyl moieties, when these are non-reducing terminal groups.

Conclusions and general significance

The present study reveals that although the activity of Abfs on artificial substrates can be quite different from that displayed on natural substrates, enzyme specificity is well conserved. This implies that carefully chosen artificial substrates bearing di-arabinofuranosyl d-xylosyl moieties are convenient tools to probe selectivity in new Abfs. Moreover, this study has further clarified the relative promiscuity of GH51 Abfs, which can apparently hydrolyze terminal disubstitutions in AXOS, albeit less efficiently than mono-substituted motifs.     

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.