l-norleucine on high glucose-induced insulin sensitivity and mitochondrial function in skeletal muscle cells.

l-norleucine, an isomer of leucine, stimulates the anabolic process of insulin. However, it is not known if and how it improves insulin sensitivity and insulin resistance. This experiment describes the generation of an insulin resistance model using high glucose-induced cells and the administration of 1.0 mmol/L l-norleucine for 48 h, to observe the effects on metabolism and gene expression in skeletal muscle cells. The results showed that l-norleucine significantly increased mitochondrial ATP content, decreased the amount of reactive oxygen species (ROS) and promoted the expression of mitochondrial generation-related genes TFAM, AMPK, PGC-1α in cells under high glucose treatment; at the same time, l-norleucine also increased glucose uptake, suggesting that l-norleucine increased insulin sensitivity and improved insulin resistance. This study suggesting that l-norleucine improves insulin resistance by ameliorating oxidative stress damage of mitochondria, improving mitochondrial function, and improving insulin sensitivity in skeletal muscle cell caused by high glucose, rather than by altering mitochondrial efficiency.

Utilization of Free and Dipeptide-Bound Formyline and Pyrraline by Saccharomyces Yeasts.

The utilization of the glycated amino acids formyline and pyrraline as well as their peptide-bound derivatives by 14 Saccharomyces yeasts, including 6 beer yeasts (bottom and top fermenting), one wine yeast, 6 strains isolated from natural habitats and one laboratory reference yeast strain (wild type) was investigated. All yeasts were able to metabolize glycated amino acids via the Ehrlich pathway to the corresponding Ehrlich metabolites. While formyline and small amounts of pyrraline entered the yeast cells via passive diffusion, the amounts of dipeptide-bound MRPs, especially the dipeptides glycated at the C-terminus, decreased much faster, indicating an uptake into the yeast cells. Furthermore, the glycation-mediated hydrophobization in general leads to an faster degradation rate compared to the native lysine dipeptides. While the utilization of free formyline is yeast-specific, the amounts of (glycated) dipeptides decreased faster in the presence of brewer's yeasts, which also showed a higher formation rate of Ehrlich metabolites compared to naturally isolated strains. Due to rapid uptake of alanyl dipeptides, it can be assumed that the Ehrlich enzyme system of naturally isolated yeasts is overloaded and the intracellularly released MRP is primarily excreted from the cell. This indicates adaptation of technologically used yeasts to (glycated) dipeptides as a nitrogen source.

Adaptive landscape flattening allows the design of both enzyme: Substrate binding and catalytic power.

Designed enzymes are of fundamental and technological interest. Experimental directed evolution still has significant limitations, and computational approaches are a complementary route. A designed enzyme should satisfy multiple criteria: stability, substrate binding, transition state binding. Such multi-objective design is computationally challenging. Two recent studies used adaptive importance sampling Monte Carlo to redesign proteins for ligand binding. By first flattening the energy landscape of the apo protein, they obtained positive design for the bound state and negative design for the unbound. We have now extended the method to design an enzyme for specific transition state binding, i.e., for its catalytic power. We considered methionyl-tRNA synthetase (MetRS), which attaches methionine (Met) to its cognate tRNA, establishing codon identity. Previously, MetRS and other synthetases have been redesigned by experimental directed evolution to accept noncanonical amino acids as substrates, leading to genetic code expansion. Here, we have redesigned MetRS computationally to bind several ligands: the Met analog azidonorleucine, methionyl-adenylate (MetAMP), and the activated ligands that form the transition state for MetAMP production. Enzyme mutants known to have azidonorleucine activity were recovered by the design calculations, and 17 mutants predicted to bind MetAMP were characterized experimentally and all found to be active. Mutants predicted to have low activation free energies for MetAMP production were found to be active and the predicted reaction rates agreed well with the experimental values. We suggest the present method should become the paradigm for computational enzyme design.

Transformation of Free and Dipeptide-Bound Glycated Amino Acids by Two Strains of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae transforms branched-chain and aromatic amino acids into higher alcohols in the Ehrlich pathway. During microbiological culturing and industrial fermentations, this yeast is confronted with amino acids modified by reducing sugars in the Maillard reaction (glycation). In order to gain some preliminary insight into the physiological "handling" of glycated amino acids by yeasts, individual Maillard reaction products (MRPs: fructosyllysine, carboxymethyllysine, pyrraline, formyline, maltosine, methylglyoxal-derived hydroimidazolone) were administered to two strains of S. cerevisiae in a rich medium. Only formyline was converted into the corresponding α-hydroxy acid, to a small extent (10 %). Dipeptide-bound pyrraline and maltosine were removed from the medium with concomitant emergence of several metabolites. Pyrraline was mainly converted into the corresponding Ehrlich alcohol (20-60 %) and maltosine into the corresponding α-hydroxy acid (40-60 %). Five specific metabolites of glycated amino acids were synthesized and characterized. We show for the first time that S. cerevisiae can use glycated amino acids as a nitrogen source and transform them into new metabolites, provided that the substances can be transported across the cell membrane.

Crystal structure of mutant form Cys115His of Citrobacter freundii methionine γ-lyase complexed with l-norleucine.

The mutant form of Citrobacter freundii methionine γ-lyase with the replacement of active site Cys115 for His has been found to be inactive in the γ-elimination reaction of methionine while fully active in the γ-elimination reaction of O-acetyl-l-homoserine and in the ß-elimination reaction of S-alk(en)yl-substituted cysteines. In this work, the crystal structure of the mutant enzyme complexed with competitive inhibitor, l-norleucine was determined at 1.45Å resolution. At the enzyme active site the inhibitor proved to be bound both noncovalently and covalently, which corresponds to the two intermediates of the γ- and ß-elimination reactions, Michaelis complex and the external aldimine. Analysis of the structure allowed us to suggest the possible reason for the inability of the mutant enzyme to catalyze the physiological reaction.

Evaluation of genetic toxicity of 6-diazo-5-oxo-l-norleucine (DON).

DON (6-diazo-5-oxo-l-norleucine), a glutamine antagonist, was demonstrated to exhibit analgesic, antibacterial, antiviral and anticancer properties. The study was performed to characterize its in vitro and in vivo genetic toxicity potential. DON was tested in the bacterial reverse mutation assay (Ames test) using Salmonella typhimurium tester strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli tester strain (WP2 uvrA) with and without S9 and also with reductive S9. In addition, DON was tested for the chromosome aberrations in Chinese hamster ovary (CHO) cells with or without S9 to evaluate the clastogenic potential. Furthermore, DON was also evaluated for its in vivo clastogenic activity by detecting micronuclei in polychromatic erythrocyte (PCE) cells in bone marrow collected from the male mice dosed intravenously with 500, 100, 10, 1 and 0.1 mg/kg at 24 and 48-h post-dose. The Ames mutagenicity assay showed no positive mutagenic responses. However, the in vitro chromosome aberration assay demonstrated dose dependent statistically positive increase in structural aberrations at 4 and 20-h exposure without S9 and also at 4-h exposure with S9. The in vivo micronucleus assay also revealed a statistically positive response for micronucleus formation at 500, 100 and 10 mg/kg at 24 and 48-h post-dose. Thus, DON appears to be negative in the Ames test but positive in the in vitro chromosome aberration assay and in the in vivo micronucleus assay. In conclusion, the results indicate DON is a genotoxic compound with a plausible epigenetic mechanism.

Semisynthetic UbH2A reveals different activities of deubiquitinases and inhibitory effects of H2A K119 ubiquitination on H3K36 methylation in mononucleosomes.

Using a genetically incorporated azidonorleucine for ubiquitin installation, we prepared multi-milligram quantities of H2AK119ub (ubH2A). With a native isopeptide linkage, the synthetic ubH2A was used to study the activity of deubiquitinases and crosstalk between H2A ubiquitination and H3K36 methylation in the context of chemically defined mononucleosomes.

Determination of Free-Form and Peptide Bound Pyrraline in the Commercial Drinks Enriched with Different Protein Hydrolysates.

Pyrraline, a causative factor for the recent epidemics of diabetes and cardiovascular disease, is also employed as an indicator to evaluate heat damage and formation of advanced glycation end-products (AGEs) in foods. Peptide-enriched drinks (PEDs) are broadly consumed worldwide due to rapid rate of absorption and perceived health effects. It can be hypothesized that PED is an important source of pyrraline, especially peptide bound pyrraline (Pep-Pyr). In this study we determined free-form pyrraline (Free-Pyr) and Pep-Pyr in drinks enriched with whey protein hydrolysate (WPH), soy protein hydrolysate (SPH) and collagen protein hydrolysate (CPH). A detection method was developed using ultrahigh-performance liquid chromatography with UV-visible detector coupled with tandem mass spectrometry after solid-phase extraction (SPE). The SPE led to excellent recovery rates ranging between 93.2% and 98.5% and a high reproducibility with relative standard deviations (RSD) of <5%. The limits of detection and quantification obtained were 30.4 and 70.3 ng/mL, respectively. Pep-Pyr was identified as the most abundant form (above 96 percent) of total pyrraline, whereas Free-Pyr was present in a small proportion (less than four percent) of total pyrraline. The results indicate that PED is an important extrinsic source of pyrraline, especially Pep-Pyr. As compared with CPH- and SPH-enriched drinks, WPH-enriched drinks contained high content of Pep-Pyr. The Pep-Pyr content is associated with the distribution of peptide lengths and the amino acid compositions of protein in PEDs.

Investigation into the misincorporation of norleucine into a recombinant protein vaccine candidate.

A high level of norleucine misincorporation was detected in a recombinant methionine-rich protein vaccine candidate expressed in E. coli K12. An investigation was conducted to evaluate a simple remediation strategy to reduce norleucine misincorporation and to determine if the phenomenon was either (a) due to the depletion of methionine during fermentation, (b) a result of the cultivation environment, or (c) a strain-specific effect. While supplementation with exogenous methionine improved product quality, the undesirable biosynthesis of non-standard amino acids such as norleucine and norvaline persisted. In contrast, non-standard amino acid biosynthesis was quickly minimized upon selection of an appropriate fed-batch process control strategy, fermentation medium, and nutrient feed. By expressing the same protein in E. coli BL21(DE3), it was determined that the biosynthesis of norleucine and norvaline, and the misincorporation of norleucine into the protein were primarily attributed to the use of E. coli K12 as the host for protein expression.

Site-specific inhibitory mechanism for amyloid ß42 aggregation by catechol-type flavonoids targeting the Lys residues.

The aggregation of the 42-residue amyloid ß-protein (Aß42) is involved in the pathogenesis of Alzheimer disease (AD). Numerous flavonoids exhibit inhibitory activity against Aß42 aggregation, but their mechanism remains unclear in the molecular level. Here we propose the site-specific inhibitory mechanism of (+)-taxifolin, a catechol-type flavonoid, whose 3',4'-dihydroxyl groups of the B-ring plays a critical role. Addition of sodium periodate, an oxidant, strengthened suppression of Aß42 aggregation by (+)-taxifolin, whereas no inhibition was observed under anaerobic conditions, suggesting the inhibition to be associated with the oxidation to form o-quinone. Because formation of the Aß42-taxifolin adduct was suggested by mass spectrometry, Aß42 mutants substituted at Arg(5), Lys(16), and/or Lys(28) with norleucine (Nle) were prepared to identify the residues involved in the conjugate formation. (+)-Taxifolin did not suppress the aggregation of Aß42 mutants at Lys(16) and/or Lys(28) except for the mutant at Arg(5). In addition, the aggregation of Aß42 was inhibited by other catechol-type flavonoids, whereas that of K16Nle-Aß42 was not. In contrast, some non-catechol-type flavonoids suppressed the aggregation of K16Nle-Aß42 as well as Aß42. Furthermore, interaction of (+)-taxifolin with the ß-sheet region in Aß42 was not observed using solid-state NMR unlike curcumin of the non-catechol-type. These results demonstrate that catechol-type flavonoids could specifically suppress Aß42 aggregation by targeting Lys residues. Although the anti-AD activity of flavonoids has been ascribed to their antioxidative activity, the mechanism that the o-quinone reacts with Lys residues of Aß42 might be more intrinsic. The Lys residues could be targets for Alzheimer disease therapy.

The Maillard reaction and food allergies: is there a link?

Food allergies are abnormal responses to a food triggered by the immune system. The majority of allergenic foods are often subjected to thermal processing before consumption. The Maillard reaction is a non-enzymatic reaction between reducing sugars and compounds with free amino groups such as amino acids and proteins, and takes place during thermal processing and storage of foods. Among many other effects the reaction leads to modification of proteins with various types of glycation structures such as Nε-(carboxymethyl-)lysine (CML), pentosidine, pyrraline and methylglyoxal-H1, which are collectively called advanced glycation end-products (AGEs). Notably, evidence has accumulated that some glycation structures of AGEs function as immune epitopes. Here we discuss the possible involvement of food allergen AGEs in the pathogenesis of food allergies.

Simultaneous analysis of the non-canonical amino acids norleucine and norvaline in biopharmaceutical-related fermentation processes by a new ultra-high performance liquid chromatography approach.

In this study, a precise and reliable ultra-high performance liquid chromatography (UHPLC) method for the simultaneous determination of non-canonical (norvaline and norleucine) and standard amino acids (aspartic acid, glutamic acid, serine, histidine, glycine, threonine, arginine, tyrosine, methionine, valine, phenylalanine, isoleucine, leucine) in biopharmaceutical-related fermentation processes was established. After pre-column derivatization with ortho-phthaldialdehyde and 2-mercaptoethanol, the derivatives were separated on a sub-2 µm particle C18 reverse-phase column. Identification and quantification of amino acids were carried out by fluorescence detection. To test method feasibility on standard HPLC instruments, the assay was properly transferred to a core-shell particle C18 reverse-phase column. The limits of detection showed excellent sensitivity by values from 0.06 to 0.17 pmol per injection and limits of quantification between 0.19 and 0.89 pmol. In the present study, the newly established UHPLC method was applied to a recombinant antibody Escherichia coli fermentation process for the analysis of total free amino acids. We were able to specifically detect and quantify the unfavorable amino acids in such complex samples. Since we observed trace amounts of norvaline and norleucine during all fermentation phases, an obligatory process monitoring should be considered to improve quality of recombinant protein drugs in future.

Malondialdehyde and 4-hydroxynonenal adducts are not formed on cardiac ryanodine receptor (RyR2) and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) in diabetes.

Recently, we reported an elevated level of glucose-generated carbonyl adducts on cardiac ryanodine receptor (RyR2) and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) in hearts of streptozotocin(STZ)-induced diabetic rats. We also showed these adduct impaired RyR2 and SERCA2 activities, and altered evoked Ca(2+) transients. What is less clear is if lipid-derived malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) also chemically react with and impair RyR2 and SERCA2 activities in diabetes? This study used western blot assays with adduct-specific antibodies and confocal microscopy to assess levels of MDA, 4-HNE, N (ε)-carboxy(methyl)lysine (CML), pentosidine, and pyrraline adducts on RyR2 and SERCA2 and evoked intracellular transient Ca(2+) kinetics in myocytes from control, diabetic, and treated-diabetic rats. MDA and 4-HNE adducts were not detected on RyR2 and SERCA2 from either control or 8 weeks diabetic rats with altered evoked Ca(2+) transients. However, CML, pentosidine, and pyrraline adducts were elevated three- to five-fold (p < 0.05). Treating diabetic rats with pyridoxamine (a scavenger of reactive carbonyl species, RCS) or aminoguanidine (a mixed reactive oxygen species-RCS scavenger) reduced CML, pentosidine, and pyrraline adducts on RyR2 and SERCA2 and blunted SR Ca(2+) cycling changes. Treating diabetic rats with the superoxide dismutase mimetic tempol had no impact on MDA and 4-HNE adducts on RyR2 and SERCA2, and on SR Ca(2+) cycling. From these data we conclude that lipid-derived MDA and 4-HNE adducts are not formed on RyR2 and SERCA2 in this model of diabetes, and are therefore unlikely to be directly contributing to the SR Ca(2+) dysregulation.

Trace element associated reduction of norleucine and norvaline accumulation during oxygen limitation in a recombinant Escherichia coli fermentation.

BACKGROUND: Norleucine and norvaline belong to a group of non-canonical amino acids which are synthesized as byproducts in the branched chain amino acid metabolism of Escherichia coli. The earlier observed misincorporation of these rare amino acids into recombinant proteins has attracted increasing attention due to the rising use of protein based biopharmaceuticals in clinical application. Experimental data revealed pyruvate overflow inducing conditions, which typically occur in oxygen limited zones of large-scale fermentations as a major reason leading to norvaline and norleucine synthesis during E. coli cultivation. Previous approaches to suppress misincorporation of norleucine and norvaline considered growth media supplementation with the relevant canonical isostructural compounds, but no research was performed on the impact of the overflow metabolism related trace elements molybdenum, nickel and selenium. These elements form essential parts of the formate hydrogen lyase (FHL) metalloprotein complex, which is a key enzyme of anaerobic pyruvate metabolism in E. coli and could therefore represent a crucial connection to the pyruvate accumulation associated biosynthesis of rare amino acids. RESULTS: In this study, the trace element associated response of recombinant antibody producing E. coli to oxygen limitation at high glucose concentration with a special focus on non-canonical amino acids was analysed. During fed-batch cultivation with provoked oxygen limitation and glucose excess norleucine and norvaline were only accumulated in the absence of molybdenum, nickel and selenium. In contrast, the trace element supplemented stress fermentation showed significantly reduced concentrations of these rare amino acids and the major signature fermentation product formate, supporting the correlation between a functional formate hydrogen lyase complex and low unspecific amino acid synthesis under oxygen limitation at high glucose concentration. CONCLUSIONS: The formation of norleucine and norvaline by recombinant E. coli during cultivation with provoked oxygen limitation and glucose excess can be reduced to levels at the detection limit by adding the trace elements molybdenum, selenium and nickel to the fermentation medium. Even under the metabolic burden during induction phase the physiologically available concentrations of non-canonical amino acids remained low. Since our results allow facile process changes that can be easily implemented to avoid the undesirable accumulation of norleucine and norvaline, we consider this study highly interesting for improved process development in E. coli based recombinant drug production and the future development of possible mechanisms to reduce misincorporation events into protein based biopharmaceuticals.

L-leucine 5-hydroxylase of Nostoc punctiforme is a novel type of Fe(II)/α-ketoglutarate-dependent dioxygenase that is useful as a biocatalyst.

L-Leucine 5-hydroxylase (LdoA) previously found in Nostoc punctiforme PCC 73102 is a novel type of Fe(II)/α-ketoglutarate-dependent dioxygenase. LdoA catalyzed regio- and stereoselective hydroxylation of L-leucine and L-norleucine into (2S,4S)-5-hydroxyleucine and (2S)-5-hydroxynorleucine, respectively. Moreover, LdoA catalyzed sulfoxidation of L-methionine and L-ethionine in the same manner as previously described L-isoleucine 4-hydroxylase. Therefore LdoA should be a promising biocatalyst for effective production of industrially useful amino acids.

Characterization of isolated yeast growth response to methionine analogs.

Methionine is one of the first limiting amino acids in poultry nutrition. The use of methionine-rich natural feed ingredients, such as soybean meal or rapeseed meal may lead to negative environmental consequences. Amino acid supplementation leads to reduced use of protein-rich ingredients. The objectives of this study were isolation of potentially high content methionine-containing yeasts, quantification of methionine content in yeasts and their respective growth response to methionine analogs. Minimal medium was used as the selection medium and the isolation medium of methionine-producing yeasts from yeast collection and environmental samples, respectively. Two yeasts previously collected along with six additional strains isolated from Caucasian kefir grains, air-trapped, cantaloupe, and three soil samples could grow on minimal medium. Only two of the newly isolated strains, K1 and C1, grew in minimal medium supplied with either methionine analogs ethionine or norleucine at 0.5% (w/v). Based on large subunit rRNA sequences, these isolated strains were identified as Pichia udriavzevii/Issatchenkia orientalis. P. kudriavzevii/I. orentalis is a generally recognized as a safe organism. In addition, methionine produced by K1 and C1 yeast hydrolysate yielded 1.3 ± 0.01 and 1.1 ± 0.01 mg g(-1) dry cell. Yeast strain K1 may be suitable as a potential source of methionine for dietary supplements in organic poultry feed but may require growth conditions to further increase their methionine content.

Interrupting the mechanisms of brain injury in a model of maple syrup urine disease encephalopathy.

Maple syrup urine disease (MSUD) was first recognized as an inherited lethal encephalopathy beginning in the first week of life and associated with an unusual odor in the urine of affected children. It was later confirmed as a deficiency of branched-chain keto acid dehydrogenase (BCKDH), which is the second step in branched-chain amino acid (BCAA) breakdown. MSUD is characterized by BCAA and branched-chain keto acid (BCKA) accumulation. BCAAs are essential amino acids and powerful metabolic signals with severe consequences of both deprivation and accumulation. Treatment requires life-long dietary restriction and monitoring of BCAAs. However, despite excellent compliance, children commonly suffer metabolic decompensation during intercurrent illness resulting in life-threatening cerebral edema and dysmyelination. The mechanisms underlying brain injury have been poorly understood. Recent studies using newly developed mouse models of both classic and intermediate MSUD have yielded insight into the consequences of rapid BCAA accumulation. Additionally, these models have been used to test preliminary treatments aimed at competing with blood-brain barrier transport of BCAA using norleucine. Assessment of biochemical changes with and without treatment suggests different roles for BCAA and BCKA in the mechanism of brain injury.

Cell-selective metabolic labeling of proteins.

Metabolic labeling of proteins with the methionine surrogate azidonorleucine can be targeted exclusively to specified cells through expression of a mutant methionyl-tRNA synthetase (MetRS). In complex cellular mixtures, proteins made in cells that express the mutant synthetase can be tagged with affinity reagents (for detection or enrichment) or fluorescent dyes (for imaging). Proteins made in cells that do not express the mutant synthetase are neither labeled nor detected.

Jadomycins derived from the assimilation and incorporation of norvaline and norleucine.

Streptomyces venezuelae ISP5230 is recognized for the production of chloramphenicol and the jadomycin family of natural products. The jadomycins are angucycline natural products containing a unique oxazolone ring incorporating an amino acid present in the minimal culture media. Substitution of different amino acids results in products of varying biological activity. Analysis of cultures of S. venezuelae ISP5230 incubated with l- and d-norvaline and l- and d-norleucine indicated that only the d-configured amino acids were incorporated into the natural products. Subsequently, jadomycin DNV and jadomycin DNL were isolated and characterized (titers 4 and 9 mg L(-1), respectively). The compounds were evaluated in the National Cancer Institute cell line cancer growth inhibition and cytotoxicity screens, for antimicrobial activity against selected Gram-positive and Gram-negative bacteria, and as DNA-cleavage agents in vitro.

The structural intolerance of the PrP alpha-fold for polar substitution of the helix-3 methionines.

The conversion of the cellular prion protein (PrP(C)) into its disease-associated form (PrP(Sc)) involves a major conformational change and the accumulation of sulfoxidized methionines. Computational and synthetic approaches have shown that this change in the polarity of M206 and M213 impacts the C-terminal domain native alpha-fold allowing the flexibility required for the structural conversion. To test the effect in the full-length molecule with site-specificity, we have generated M-to-S mutations. Molecular dynamics simulations show that the replacement indeed perturbs the native state. When this mutation is placed at the conserved methionines of HaPrP(23-231), only substitutions at the Helix-3 impair the alpha-fold, stabilizing a non-native state with perturbed secondary structure, loss of native tertiary contacts, increased surface hydrophobicity, reduced thermal stability and an enhanced tendency to aggregate into protofibrillar polymers. Our work supports that M206 and M213 function as alpha-fold gatekeepers and suggests that their redox state regulate misfolding routes.