Inhibiting the color formation by gradient temperature‐elevating Maillard reaction of soybean peptide‐xylose system based on interaction of l‐cysteine and Amadori compounds

Light color and savory flavor enhancer are attractive for consumers and food producers. The effect of addition time of l ‐cysteine on inhibiting color formation was investigated in soybean peptide‐xylose system, and the possible pathway was explored. Once dicarbonyl compounds were formed during the Maillard reaction, the addition of l ‐cysteine had no color‐inhibiting effect; if l ‐cysteine was added immediately after the Amadori compound was formed, the extraordinary color‐inhibiting effect was observed. Therefore, an improved way to inhibit color formation was proposed on the basis of the interaction of l ‐cysteine and Amadori compounds by controlling the addition time of l ‐cysteine through gradient temperature‐elevating Maillard reaction. The system was heated at 80 °C for 60 min to form Amadori compounds, followed by the addition of L‐cysteine, and the temperature was raised to 120 °C and held for 110 min. Compared with traditional products, the lightest color product was found desirable by GC/MS analysis and sensory evaluation. The novel method proposed can be a guide for the industrial preparation of light‐colored products. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Negative modulation of the GABAAρ1 receptor function by l‐cysteine

l ‐Cysteine is an endogenous sulfur‐containing amino acid with multiple and varied roles in the central nervous system, including neuroprotection and the maintenance of the redox balance. However, it was also suggested as an excitotoxic agent implicated in the pathogenesis of neurological disorders such as Parkinson′s and Alzheimer′s disease. l ‐Cysteine can modulate the activity of ionic channels, including voltage‐gated calcium channels and glutamatergic NMDA receptors, whereas its effects on GABAergic neurotransmission had not been studied before. In the present work, we analyzed the effects of l ‐cysteine on responses mediated by homomeric GABA_Aρ1 receptors, which are known for mediating tonic γ‐aminobutyric acid (GABA) responses in retinal neurons. GABA_Aρ1 receptors were expressed in Xenopus laevis oocytes and GABA‐evoked chloride currents recorded by two‐electrode voltage‐clamp in the presence or absence of l ‐cysteine. l ‐Cysteine antagonized GABA_Aρ1 receptor‐mediated responses; inhibition was dose‐dependent, reversible, voltage independent, and susceptible to GABA concentration. Concentration‐response curves for GABA were shifted to the right in the presence of l ‐cysteine without a substantial change in the maximal response. l ‐Cysteine inhibition was insensitive to chemical protection of the sulfhydryl groups of the ρ1 subunits by the irreversible alkylating agent N‐ethyl maleimide. Our results suggest that redox modulation is not involved during l ‐cysteine actions and that l ‐cysteine might be acting as a competitive antagonist of the GABA_Aρ1 receptors.

     

Nuclear magnetic resonance‐based determination of dioxygen binding sites in protein cavities

The location and ligand accessibility of internal cavities in cysteine‐free wild‐type T4 lysozyme was investigated using O₂ gas‐pressure NMR spectroscopy and molecular dynamics (MD) simulation. Upon increasing the concentration of dissolved O₂ in solvent to 8.9 mM, O₂‐induced paramagnetic relaxation enhancements (PREs) to the backbone amide and side chain methyl protons were observed, specifically around two cavities in the C‐terminal domain. To determine the number of O₂ binding sites and their atomic coordinates from the 1/r⁶ distance dependence of the PREs, we established an analytical procedure using Akaike's Information Criterion, in combination with a grid‐search. Two O₂‐accessible sites were identified in internal cavities: One site was consistent with the xenon‐binding site in the protein in crystal, and the other site was established to be a novel ligand‐binding site. MD simulations performed at 10 and 100 mM O₂ revealed dioxygen ingress and egress as well as rotational and translational motions of O₂ in the cavities. It is therefore suggested that conformational fluctuations within the ground‐state ensemble transiently develop channels for O₂ association with the internal protein cavities.     

Perturbation of the tris(2,2′‐bipyridine) ruthenium(II)‐catalyzed Belousov–Zhabotinsky oscillating chemiluminescence reaction by l‐cysteine and its application

Perturbation of the tris(2,2′‐bipyridine)ruthenium(II) [Ru(bpy)₃²⁺]‐catalyzed Belousov–Zhabotinsky (BZ) oscillating chemiluminescence (CL) reaction induced by l ‐cysteine was observed in the closed system. It was found that the CL intensity was decreased in the presence of l ‐cysteine. Meanwhile, oscillation period and oscillating induction period were prolonged. The sufficient reproducible induction period was used as parameter for the analytical application of oscillating CL reaction. Under the optimum conditions, the changes in the oscillating CL induction period were linearly proportional to the concentration of l ‐cysteine in the range from 8.0 × 10^?7 to 5.0 × 10^?5 mol L^?1 (r = 0.997) with a detection limit of 4.3 × 10^?7 mol L^?1. The possible mechanism of l ‐cysteine perturbation on the oscillating CL reaction was also discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

Crystallographic and mutational analyses of cystathionine β‐synthase in the H2S‐synthetic gene cluster in Lactobacillus plantarum

Cystathionine β‐synthase (CBS) catalyzes the formation of l ‐cystathionine from l ‐serine and l ‐homocysteine. The resulting l ‐cystathionine is decomposed into l ‐cysteine, ammonia, and α‐ketobutylic acid by cystathionine γ‐lyase (CGL). This reverse transsulfuration pathway, which is catalyzed by both enzymes, mainly occurs in eukaryotic cells. The eukaryotic CBS and CGL have recently been recognized as major physiological enzymes for the generation of hydrogen sulfide (H₂S). In some bacteria, including the plant‐derived lactic acid bacterium Lactobacillus plantarum, the CBS‐ and CGL‐encoding genes form a cluster in their genomes. Inactivation of these enzymes has been reported to suppress H₂S production in bacteria; interestingly, it has been shown that H₂S suppression increases their susceptibility to various antibiotics. In the present study, we characterized the enzymatic properties of the L. plantarum CBS, whose amino acid sequence displays a similarity with those of O‐acetyl‐l ‐serine sulfhydrylase (OASS) that catalyzes the generation of l ‐cysteine from O‐acetyl‐l ‐serine (l ‐OAS) and H₂S. The L. plantarum CBS shows l ‐OAS‐ and l ‐cysteine‐dependent CBS activities together with OASS activity. Especially, it catalyzes the formation of H₂S in the presence of l ‐cysteine and l ‐homocysteine, together with the formation of l ‐cystathionine. The high affinity toward l ‐cysteine as a first substrate and tendency to use l ‐homocysteine as a second substrate might be associated with its enzymatic ability to generate H₂S. Crystallographic and mutational analyses of CBS indicate that the Ala70 and Glu223 residues at the substrate binding pocket are important for the H₂S‐generating activity.     

Cysteine inhibits amyloid fibrillation of lysozyme and directs the formation of small worm‐like aggregates through non‐covalent interactions

In this article, we discuss the effects of amino acids on amyloid aggregation of lysozyme. l ‐cysteine (Cys) dramatically inhibited fibrillation of lysozyme, whereas other amino acids (including l ‐arginine) did not. In the presence of Cys, the aggregation pathway of lysozyme shifted from fibrillation to the formation of the small worm‐like aggregates with unfolding. The interaction between Cys and lysozyme was observed to be non‐covalent, suggesting that the thiophilic interaction between the thiol group on the side chain of Cys and the core sequence of lysozyme significantly contributes to the inhibition of amyloid aggregation. These findings provide a new basis for the design of a biocompatible additive to prevent amyloid fibrillation. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:470–478, 2014     

Nucleation temperature‐controlled synthesis and in vitro toxicity evaluation of l‐cysteine‐capped Mn:ZnS quantum dots for intracellular imaging

Quantum dots (QDs), one of the fastest developing and most exciting fluorescent materials, have attracted increasing interest in bioimaging and biomedical applications. The long‐term stability and emission in the visible region of QDs have proved their applicability as a significant fluorophore in cell labelling. In this study, an attempt has been made to explore the efficacy of l ‐cysteine as a capping agent for Mn‐doped ZnS QD for intracellular imaging. A room temperature nucleation strategy was adopted to prepare non‐toxic, water‐dispersible and biocompatible Mn:ZnS QDs. Aqueous and room temperature QDs with l ‐cysteine as a capping agent were found to be non‐toxic even at a concentration of 1500 µg/mL and have wide applications in intracellular imaging. Copyright © 2015 John Wiley & Sons, Ltd.     

Enhanced dissolution of the substrate d,l‐2‐amino‐Δ2‐thiazoline‐4‐carboxylic acid and enzymatic production of l‐cysteine at high concentrations

l ‐Cysteine is widely used as a precursor in the pharmaceutical, cosmetic, food, and feed additive industries. It has been industrially produced from hydrolysis of human and animal hairs, which is limited for industrial production. At the same time, chemical hydrolysis causes the formation of intractable waste material. Thus, environmentally friendly methods have been developed. A big obstacle of currently available methods is the low substrate solubility leading to poor l ‐cysteine yield. Here, a method for improving the low solubility of the substrate d ,l ‐2‐amino‐Δ²‐thiazoline‐4‐carboxylic acid (d ,l ‐ATC) is presented and the enzymatic reaction at high concentration levels was optimized. The substrate was dissolved in large amounts in aqueous solutions by pH control using salts. d ,l ‐ATC solubility increased with an increasing solution pH due to its enhanced hydrophilicity, which can be achieved by a shift to dissociated carboxylic group (–COO^?). The highest d ,l ‐ATC solubility of 610 mM was obtained at pH 10.5. The maximum l ‐cysteine yield of 250 mM was attained at pH 9.1, which lies between the optimum values for high substrate solubility and reaction rate. The product yield could be increased by more than 10 times compared to those in previous reports, which is industrially meaningful.     

Preventive effects of N‐acetyl‐l‐cysteine against imidacloprid intoxication on Bombyx mori larvae

Imidacloprid, a widely used neonicotinoid insecticide, is toxic to silkworm (Bombyx mori). To explore whether N‐acetyl‐l ‐cysteine (NAC) has an effect on preventing silkworm (B. mori) from toxification caused by imidacloprid, we fed the fifth‐instar larvae with mulberry leaves dipped in 200 mg/L NAC solution before exposing in imidacloprid, and investigated the silkworm growth, survival rate, feed efficiency, cocoon quality, and the activities of antioxidant enzymes in midgut. The results showed that addition of NAC could significantly increase body weight, survival rate, and feed efficiency of imidacloprid poisoned silkworm larvae (P < 0.05), as well as cocoon mass, cocoon shell mass, and the ratio of cocoon shell (P < 0.05). Furthermore, it could significantly promote the activities of the antioxidant enzymes including superoxide dismutase, catalase, and glutathione peroxide in the midgut of fifth‐instar larvae under imidacloprid exposure at the late stage of treatment. In addition, it also could downregulate the malondialdehyde content. The results of our findings proved that the added NAC may have some beneficial effects on protection or restoration of antioxidant balance in imidacloprid exposed larvae.     

Unveiling the Pathways of Dioxygen Through the C2 Component of the Environmentally Relevant Monooxygenase p‐Hydroxyphenylacetate Hydroxylase from Acinetobacter baumannii: A Molecular Dynamics Investigation

In this work, models of the homotetrameric C2 component of the monooxygenase p‐hydroxyphenylacetate hydroxylase from Acinetobacter baumannii, in complex with dioxygen (O₂) and, or not, the substrate p‐hydroxyphenylacetate (HPA) were built. Both models proved to be amenable to random‐acceleration molecular dynamics (RAMD) simulations, whereby a tiny randomly oriented external force, acting on O₂ at the active site in front of flavin mononucleotide (FMNH^?), accelerated displacement of O₂ toward the bulk solvent. This allowed us to carry out a sufficiently large number of RAMD simulations to be of statistical significance. The two systems behaved very similarly under RAMD, except for O₂ leaving the active site more easily in the absence of HPA, but then finding similar obstacles in getting to the gate as when the active site was sheltered by HPA. This challenges previous conclusions that HPA can only reach the active center after that the C4aOOH derivative of FMNH^? is formed, requiring uptake of O₂ at the active site before HPA. According to these RAMD simulations, O₂ could well get to FMNH^? also in the presence of the substrate at the active site.     

Melanin Granules Prevent the Cytotoxic Effects of l‐DOPA on Retinal Pigment Epithelial Cells in Vitro by Regulation of NO and Superoxide Radicals

Inasmuch as the nitrogen cycle elicits the direct reduction of N₂ to NH₃ through enzymatic reactions, and inasmuch as l ‐DOPA ( l ‐dihydroxyphentlalamine), a catecholamine, can be a source of nitric oxide (NO), it is possible that melanin granules in the eye affect the generation of NO, which causes damage to the retinal pigment epithelial (RPE) cells during the oxidation of l ‐DOPA. In order to confirm this possibility, we analyzed the correlations of NO generation, cell growth, and superoxide dismutase (SOD) activities in two types (melanotic and amelanotic) of bovine RPE cells following exposure to l ‐DOPA. NO generation from l ‐DOPA was determined using an NO detector that is reliant on redox currents. The concentration of NO was measured in terms of diffusion currents run between a working electrode and a counter electrode, both being set in culture medium placed in a Petri dish. For the assays, l ‐DOPA was added to the medium at various concentrations (5, 29.9, 79.4, 152.7 or 249 μM), and 6 min after addition, an NO‐trapping agent 2,4‐carboxyphenyl‐4,4,5,5‐tetramethylimidazole‐1‐oxyl 3‐oxide (carboxy‐PTIO) was also added. The melanotic and amelanotic types of RPE cells were cultured separately in medium with l ‐DOPA under an atmosphere containing 20, 10 or 5% oxygen. Cell numbers were counted using a Coulter counter, and SOD activities were determined following incubation for 24, 48 or 72 hr using a modification of the luminol assay. The results obtained indicated that: (a) NO was produced from l ‐DOPA in a concentration‐dependent manner and was trapped quantitatively by carboxy‐PTIO; (b) the generation of NO was inhibited more markedly in the melanotic cell line than in the amelanotic one, suggesting an increased tolerance to l ‐DOPA‐derived cytotoxicity in the former; and (c) the SOD activities were more affected by oxygen concentration in the melanotic cells than in the amelanotic ones. From these results, it is concluded that melanin granules in RPE cells have a role in preventing the cytotoxicity derived from l ‐DOPA and in regulating the generation of NO and superoxide radicals.     

Cysteine Suppresses Oxidative Stress-Induced Myofibrillar Proteolysis in Chick Myotubes

The effects of cysteine as an antioxidant nutrient on change in protein modification and myofibrillar proteolysis in chick myotubes by induction of oxidative stress by H₂O₂ treatment were investigated. Myotubes were treated for 1 h with H₂O₂ (1 mM). After this treatment, the H₂O₂ was removed and the cells were cultured in cysteine (0.1 and 1 mM) containing serum-free medium for 24 h. Protein carbonyl content as an index of protein modification and N^τ-methylhistidine release as an index of myofibrillar proteolysis were increased at 24 h after H₂O₂ treatment, and the increment was reduced by cysteine. Calpain, proteasome and cathepsin (B+L and D) activities were increased at 24 h after H₂O₂ treatment, and the increment was also reduced by cysteine. These results indicate that cysteine suppresses protein modification by oxidative stress, resulting in a decrease of protease acitivities, finally resulting in a decrease in myofibrillar proteolysis in chick myotubes.     

Structures of the Neisseria meningitides methionine‐binding protein MetQ in substrate‐free form and bound to l‐ and d‐methionine isomers

The bacterial periplasmic methionine‐binding protein MetQ is involved in the import of methionine by the cognate MetNI methionine ATP binding cassette (ABC) transporter. The MetNIQ system is one of the few members of the ABC importer family that has been structurally characterized in multiple conformational states. Critical missing elements in the structural analysis of MetNIQ are the structure of the substrate‐free form of MetQ, and detailing how MetQ binds multiple methionine derivatives, including both l ‐ and d ‐methionine isomers. In this study, we report the structures of the Neisseria meningitides MetQ in substrate‐free form and in complexes with l ‐methionine and with d ‐methionine, along with the associated binding constants determined by isothermal titration calorimetry. Structures of the substrate‐free (N238A) and substrate‐bound N. meningitides MetQ are related by a “Venus‐fly trap” hinge‐type movement of the two domains accompanying methionine binding and dissociation. l ‐ and d ‐methionine bind to the same site on MetQ, and this study emphasizes the important role of asparagine 238 in ligand binding and affinity. A thermodynamic analysis demonstrates that ligand‐free MetQ associates with the ATP‐bound form of MetNI ～40 times more tightly than does liganded MetQ, consistent with the necessity of dissociating methionine from MetQ for transport to occur.     

Protonation states of active‐site lysines of penicillin‐binding protein 6 from Escherichia coli and the mechanistic implications

The protonation states of the two active‐site lysines (Lys69 and Lys235) of PBP 6 of Escherichia coli were explored to understand the active site chemistry of this enzyme. Each lysine was individually mutated to cysteine, and the resultant two mutant proteins were purified to homogeneity. Each protein was denatured, and its cysteine was chemically modified to produce an S‐aminoethylated cysteine (γ‐thialysine) residue. Following renaturation, the evaluation of the kinetics of the dd ‐carboxypeptidase activity of PBP 6 as a function of pH was found consistent with one lysine in its free‐base (Lys69) and the other in the protonated state (Lys235) for optimal catalysis. The experimental estimates for their pK_a values were compared with the pK_a values calculated computationally, using molecular‐dynamics simulations and a thermodynamic cycle. Study of the γ‐thialysine69 showed that lysine at position 69 influenced the basic limb of catalysis, consistent with the fact that the two lysine side chains are in proximity to each other in the active site. Based on these observations, a reaction sequence for PBP 6 is proposed, wherein protonated Lys235 serves as the electrostatic substrate anchor and Lys69 as the conduit for protons in the course of the acylation and deacylation half‐reactions. Proteins 2014; 82:1348–1358. © 2013 Wiley Periodicals, Inc.     

Screening of l‐histidine‐based ligands to modify monolithic supports and selectively purify the supercoiled plasmid DNA isoform

The growing demand of pharmaceutical‐grade plasmid DNA (pDNA) suitable for biotherapeutic applications fostered the development of new purification strategies. The surface plasmon resonance technique was employed for a fast binding screening of l ‐histidine and its derivatives, 1‐benzyl‐l ‐histidine and 1‐methyl‐l ‐histidine, as potential ligands for the biorecognition of three plasmids with different sizes (6.05, 8.70, and 14 kbp). The binding analysis was performed with different isoforms of each plasmid (supercoiled, open circular, and linear) separately. The results revealed that the overall affinity of plasmids to l ‐histidine and its derivatives was high (K_D > 10⁻⁸ M), and the highest affinity was found for human papillomavirus 16 E6/E7 (K_D = 1.1 × 10⁻¹⁰ M and K_D = 3.34 × 10⁻¹⁰ M for open circular and linear plasmid isoforms, respectively). l ‐Histidine and 1‐benzyl‐l ‐histidine were immobilized on monolithic matrices. Chromatographic studies of l ‐histidine and 1‐benzyl‐l ‐histidine monoliths were also performed with the aforementioned samples. In general, the supercoiled isoform had strong interactions with both supports. The separation of plasmid isoforms was achieved by decreasing the ammonium sulfate concentration in the eluent, in both supports, but a lower salt concentration was required in the 1‐benzyl‐l ‐histidine monolith because of stronger interactions promoted with pDNA. The efficiency of plasmid isoforms separation remained unchanged with flow rate variations. The binding capacity for pDNA achieved with the l ‐histidine monolith was 29‐fold higher than that obtained with conventional l ‐histidine agarose. Overall, the combination of either l ‐histidine or its derivatives with monolithic supports can be a promising strategy to purify the supercoiled isoform from different plasmids with suitable purity degree for pharmaceutical applications. Copyright © 2015 John Wiley & Sons, Ltd.     

On the Pathways of Biologically Relevant Diatomic Gases through Proteins. Dioxygen and Heme Oxygenase from the Perspective of Molecular Dynamics

This work deals with dioxygen (O₂) binding sites and pathways through inducible human heme oxygenase (HO‐1). The experimentally known distal binding site 1, and sites 2–3 above it, could be reproduced by means of non‐deterministic random‐acceleration molecular‐dynamics (RAMD) simulations. In addition, RAMD revealed the proximal binding site 5, a deeply‐seated binding site 4, which lies behind heme, as well as a few gates communicating with the external medium. In getting from site 1 to the main gate, which lies on the protein front opposed to site 4, O₂ follows chiefly the shortest direct pathway. Less frequently, O₂ visits intermediate sites 2, 4, or 5 along longer pathways. A similarity between HO‐1, myoglobin, and cytoglobin in using, for diatomic gas delivery, the direct shortest pathway from the heme center to the surrounding medium, is emphasized. Otherwise, comparing other proteins and diatomic gases, each system reveals its peculiarities as to sites, gates, and pathways. Thus, relating these properties to the physiological functions of the proteins remains in general a challenge for future studies.     

Geometric considerations support the double‐displacement catalytic mechanism of l‐asparaginase

Twenty crystal structures of the complexes of l ‐asparaginase with l ‐Asn, l ‐Asp, and succinic acid that are currently available in the Protein Data Bank, as well as 11 additional structures determined in the course of this project, were analyzed in order to establish the level of conservation of the geometric parameters describing interactions between the substrates and the active site of the enzymes. We found that such stereochemical relationships are highly conserved, regardless of the organism from which the enzyme was isolated, specific crystallization conditions, or the nature of the ligands. Analysis of the geometry of the interactions, including Bürgi–Dunitz and Flippin–Lodge angles, indicated that Thr12 (Escherichia coli asparaginase II numbering) is optimally placed to be the primary nucleophile in the most likely scenario utilizing a double‐displacement mechanism, whereas catalysis through a single‐displacement mechanism appears to be the least likely.     

Identification of a flavin‐containing S‐oxygenating monooxygenase involved in alliin biosynthesis in garlic

S‐Alk(en)yl‐l ‐cysteine sulfoxides are cysteine‐derived secondary metabolites highly accumulated in the genus Allium. Despite pharmaceutical importance, the enzymes that contribute to the biosynthesis of S‐alk‐(en)yl‐l ‐cysteine sulfoxides in Allium plants remain largely unknown. Here, we report the identification of a flavin‐containing monooxygenase, AsFMO1, in garlic (Allium sativum), which is responsible for the S‐oxygenation reaction in the biosynthesis of S‐allyl‐l ‐cysteine sulfoxide (alliin). Recombinant AsFMO1 protein catalyzed the stereoselective S‐oxygenation of S‐allyl‐l ‐cysteine to nearly exclusively yield (R_CS_S)‐S‐allylcysteine sulfoxide, which has identical stereochemistry to the major natural form of alliin in garlic. The S‐oxygenation reaction catalyzed by AsFMO1 was dependent on the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and flavin adenine dinucleotide (FAD), consistent with other known flavin‐containing monooxygenases. AsFMO1 preferred S‐allyl‐l ‐cysteine to γ‐glutamyl‐S‐allyl‐l ‐cysteine as the S‐oxygenation substrate, suggesting that in garlic, the S‐oxygenation of alliin biosynthetic intermediates primarily occurs after deglutamylation. The transient expression of green fluorescent protein (GFP) fusion proteins indicated that AsFMO1 is localized in the cytosol. AsFMO1 mRNA was accumulated in storage leaves of pre‐emergent nearly sprouting bulbs, and in various tissues of sprouted bulbs with green foliage leaves. Taken together, our results suggest that AsFMO1 functions as an S‐allyl‐l ‐cysteine S‐oxygenase, and contributes to the production of alliin both through the conversion of stored γ‐glutamyl‐S‐allyl‐l ‐cysteine to alliin in storage leaves during sprouting and through the de novo biosynthesis of alliin in green foliage leaves.