Preparation of 3-bromo-l-tyrosine and 3,5-dibromo-l-tyrosine

l-Tyrosine is converted to 3-bromo-l-tyrosine in good yield by reaction with 1.2 equiv. of DMSO in HBr/AcOH, while reaction with 2.2 equiv. of DMSO under comparable conditions results in formation of 3,5-dibromo-l-tyrosine in good yield. This is the simplest, safest and most efficient method for the preparation of gram quantities of either 3-bromo-l-tyrosine or 3,5-dibromo-l-tyrosine.     

Phenylalanine hydroxylase activity in a mutant of Geotrichum candidum

A mutant of Geotrichum candidum was isolated with a tyrosine requirement which could be satisfied by l-tyrosine or l-phenylalanine. l-Phenylalanine is converted by cell suspensions to l-tyrosine, which can be detected in the growth medium. The incorporation of the tyrosine into cell protein is described. l-Phenylalanine is converted to tyrosine by cell-free extracts with a requirement for some dialysable components. The adaptation of intact cells to phenylalanine metabolism is also described.     

Optimization of Biotransformation of l-Tyrosine to l-DOPA by Yarrowia lipolytica-NCIM 3472 Using Response Surface Methodology

l-DOPA (3,4-dihydroxyphenyl-l-alanine) is the most widely used drug for treatment of Parkinson’s disease. In this study Yarrowia lipolytica-NCIM 3472 biomass was used for transformation of l-tyrosine to l-DOPA. The process parameters were optimized using response surface methodology (RSM). The optimum values of the tested variables for the production of l-DOPA were: pH 7.31, temperature 42.9 °C, 2.31 g l^?1 cell mass and 1.488 g l^?1 l-tyrosine. The highest yield obtained with these optimum parameters along with recycling of the cells was 4.091 g l^?1. This optimization of process parameters using RSM resulted in 4.609-fold increase in the l-DOPA production. The statistical analysis showed that the model was significant. Also coefficient of determination (R²) was 0.9758, indicating a good agreement between the experimental and predicted values of l-DOPA production. The highest tyrosinase activity observed was 7,028 U mg^?1 tyrosine. l-DOPA production was confirmed by HPTLC and HPLC analysis. Thus, RSM approach effectively enhanced the potential of Y. lipolytica-NCIM 3472 as an alternative source to produce l-DOPA.     

l-Tyrosine Induces DNA Damage in Brain and Blood of Rats

Mutations in the tyrosine aminotransferase gene have been identified to cause tyrosinemia type II which is inherited in an autosomal recessive manner. Studies have demonstrated that an excessive production of ROS can lead to reactions with macromolecules, such as DNA, lipids, and proteins. Considering that the l-tyrosine may promote oxidative stress, the main objective of this study was to investigate the in vivo effects of l-tyrosine on DNA damage determined by the alkaline comet assay, in brain and blood of rats. In our acute protocol, Wistar rats (30 days old) were killed 1 h after a single intraperitoneal l-tyrosine injection (500 mg/kg) or saline. For chronic administration, the animals received two subcutaneous injections of l-tyrosine (500 mg/kg, 12-h intervals) or saline administered for 24 days starting at postnatal day (PD) 7 (last injection at PD 31), 12 h after the last injection, the animals were killed by decapitation. We observed that acute administration of l-tyrosine increased DNA damage frequency and damage index in cerebral cortex and blood when compared to control group. Moreover, we observed that chronic administration of l-tyrosine increased DNA damage frequency and damage index in hippocampus, striatum, cerebral cortex and blood when compared to control group. In conclusion, the present work demonstrated that DNA damage can be encountered in brain from animal models of hypertyrosinemia, DNA alterations may represent a further means to explain neurological dysfunction in this inherited metabolic disorder and to reinforce the role of oxidative stress in the pathophysiology of tyrosinemia type II.     

The effect of some environmental factors on the production of L-DOPA by alginate-entrapped cells of Mucuna pruriens

In-vitro-grown cells of Mucuna pruriens, immobilized in calcium-alginate gels, were able to transform the precursor L-tyrosine into L-dihydroxyphenylalanine (L-DOPA). After the immobilization in alginate the plant cells released 90% of the produced L-DOPA into the medium; supplementation of the medium with calcium inhibited both the transformation of L-tyrosine into L-DOPA and the release of L-DOPA into the medium. Continuous illumination of the beads had a slight beneficial effect on the synthesis of L-DOPA. A simple production medium for the transformation of L-tyrosine into L-DOPA was designed. This medium contained only sucrose and sodium chloride as osmotic stabilizers, a low concentration of calcium chloride for stabilization of the alginate beads, and L-tyrosine as the precursor.     

Effect of Acute and Chronic Administration of l-Tyrosine on Nerve Growth Factor Levels in Rat Brain

Most inborn errors of tyrosine catabolism produce hypertyrosinemia. Neurological manifestations are variable and some patients are developmentally normal, while others show different degrees of developmental retardation. Considering that current data do not eliminate the possibility that elevated levels of tyrosine and/or its derivatives may have noxious effects on central nervous system development in some patients, the present study evaluated nerve growth factor (NGF) levels in hippocampus, striatum and posterior cortex of young rats. In our acute protocol, Wistar rats (10 and 30 days old) were killed 1 h after a single intraperitoneal administration of l-tyrosine (500 mg/kg) or saline. Chronic administration consisted of l-tyrosine (500 mg/kg) or saline injections 12 h apart for 24 days in Wistar rats (7 days old); the rats were killed 12 h after the last injection. NGF levels were then evaluated. Our findings showed that acute administration of l-tyrosine decreased NGF levels in striatum of 10-day-old rats. In the 30-day-old rats, NGF levels were decreased in hippocampus and posterior cortex. On the other hand, chronic administration of l-tyrosine increased NGF levels in posterior cortex. Decreased NGF may impair growth, differentiation, survival and maintenance of neurons.     

Enantiomer-specific selection of amino acids

Dietary intake of l-amino acids impacts on several physiological functions, including the control of gastrointestinal motility, pancreatic secretion, and appetite. However, the biological mechanisms regulating behavioral predilections for certain amino acid types remain poorly understood. We tested the hypothesis that, in mice, the potency with which a given glucogenic amino acid increases glucose utilization reflects its rewarding properties. We have found that: (1) during long-, but not short-, term preference tests, l-alanine and l-serine were preferred over their d-enantiomer counterparts, while no such effect was observed for l-threonine vs. d-threonine; (2) these behavioral patterns were closely associated with the ability of l-amino acids to promote increases in respiratory exchange ratios such that those, and only those, l-amino acids able to promote increases in respiratory exchange ratios were preferred over their d-isomers; (3) these behavioral preferences were independent of gustatory influences, since taste-deficient Trpm5 knockout mice displayed ingestive responses very similar to those of their wild-type counterparts. We conclude that the ability to promote increases in respiratory exchange ratios enhances the reward value of nutritionally relevant amino acids and suggest a mechanistic link between substrate utilization and amino acid preferences.     

Laccase-catalyzed cross-linking of amino acids and peptides with dihydroxylated aromatic compounds

In order to design potential biomaterials, we investigated the laccase-catalyzed cross-linking between l-lysine or lysine-containing peptides and dihydroxylated aromatics. l-Lysine is one of the major components of naturally occurring mussel adhesive proteins (MAPs). Dihydroxylated aromatics are structurally related to 3,4-dihydroxyphenyl-l-alanine, another main component of MAPs. Mass spectrometry and nuclear magnetic resonance analyses show that the ε-amino group of l-lysine is able to cross-link dihydroxylated aromatics. Additional oligomer and polymer cross-linked products were obtained from di- and oligopeptides containing l-lysine. Potential applications in medicine or industry for biomaterials synthesised via the three component system consisting of the oligopeptide [Tyr-Lys]₁₀, dihydroxylated aromatics and laccase are discussed.     

Cyclic tetrapeptides with –SS– bridging between amino acid side chains for potent histone deacetylases’ inhibition

Cyclic depsipeptide FK228 with an intramolecular disulfide bond is a potent inhibitor of histone deacetylases (HDAC). FK228 is stable in blood because of its prodrug function, whose –SS– bond is reduced within the cell. Here, cyclic peptides with –SS– bridges between a variety of amino acids were synthesized and assayed for HDAC inhibition. Cyclic peptide 3, cyclo(-l-amino acid-l-amino acid-l-Val-d-Pro-), with an –SS– bridge between the first and second amino acids, was found to be a potent HDAC inhibitor. Cyclic peptide 7, cyclo(-l-amino acid-d-amino acid-l-Val-d-Pro-), with an –SS– bridge between the first and second amino acids, was also a potent HDAC inhibitor.     

Characterization of ergothionase from Burkholderia sp. HME13 and its application to enzymatic quantification of ergothioneine

We identified ergothionase, which catalyzes conversion of ergothioneine to thiolurocanic acid and trimethylamine, in a newly isolated ergothioneine-utilizing strain, Burkholderia sp. HME13. The enzyme was purified and its N-terminal amino acid sequence was determined. Based on the amino acid sequence, the gene encoding the enzyme was cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity and characterized. The enzyme consisted of four identical 55-kDa subunits. The enzyme showed maximum activity at pH 8.0 and 65 °C and was stable between pH 7.0 and pH 10.0 and up to 60 °C. The enzyme acted on ergothioneine (K _m: 19 μM, V _max: 270 μmol/min/mg), but not d-histidine, l-histidine, d-tyrosine, l-tyrosine, d-phenylalanine, or l-phenylalanine. The enzyme was activated by BaCl₂ and strongly inhibited by CuSO₄, ZnSO₄, and HgCl₂. The amino acid sequence of ergothionase showed 23 % similarity to histidine ammonia-lyase (HAL) from Pseudomonas putida and 17 % similarity to phenylalanine ammonia-lyase (PAL) from parsley. However, the tripeptide sequence, Ala-Ser-Gly, which is important for catalysis in both HAL and PAL, was not conserved in ergothionase. The application of ergothionase for the quantification of ergothioneine contained in practical food and blood samples was investigated by performing a recovery test. Satisfactory recovery data (98.7–104 %) were obtained when ergothioneine was added to extract of tamogitake and hemolysis blood.     

Development of a two-stage feeding strategy based on the kind and level of feeding nutrients for improving fed-batch production of l-threonine by Escherichia coli

Fed-batch fermentation is the predominant method for industrial production of amino acids. In this study, we comprehensively investigated the effects of four kinds of feeding nutrients and developed an accurate optimization strategy for fed-batch production of l-threonine. The production of l-threonine was severely inhibited when cell growth ceased in the bath culture. Similarly, l-threonine production was also associated with cell growth in the carbon-, phosphate-, and sulfate-limited fed-batch cultures, but the accumulation of l-threonine was markedly increased because of the extended production time in the growth stage. Interestingly, auxotrophic amino acid (l-isoleucine)-limited feeding promoted l-threonine production over the non-growth phase. Metabolite analysis indicates that substantial production of acetate and glutamate and the resulting accumulation of ammonium may lead to the inhibition of l-threonine production. During the growth phase, the levels of l-isoleucine were accurately optimized by balancing cell growth and production with Pontryagin’s maximum principle, basing on the relationship between the specific growth rate μ and specific production rate ρ. Furthermore, the depletion of l-isoleucine and phosphate at the end of the growth phase favored the synthesis of l-threonine in the subsequent non-growth phase. Combining the two-stage feeding profiles, the final l-threonine concentration and conversion rate were increased by 5.9- and 2.1-fold, respectively, compared to batch processes without feeding control. The identification of efficient feeding nutrient and the development of accurate feeding strategies provide potential guidelines for microbial production of amino acids.     

Probing the specificity of gamma-glutamylamine cyclotransferase: an enzyme involved in the metabolism of transglutaminase-catalyzed protein crosslinks

γ-Glutamylamine cyclotransferase (gGACT) catalyzes the intramolecular cyclization of a variety of l-γ-glutamylamines producing 5-oxo-l-proline and free amines. Its substrate specificity implicates it in the downstream metabolism of transglutaminase products, and is distinct from that of γ-glutamyl cyclotransferase which acts on l-γ-glutamyl amino acids. To elucidate the mechanism by which gGACT distinguishes between l-γ-glutamylamine and amino acid substrates, the specificity of the rabbit kidney enzyme for the amide region of substrates was probed through the kinetic analysis of a series of l-γ-glutamylamines. The isodipeptide N ^?-(l-γ-glutamyl)-l-lysine 1 was used as a reference. The kinetic constants of the l-γ-glutamyl derivative of n-butylamine 7, were nearly identical to those of 1. Introduction of a methyl or carboxylate group on the carbon adjacent to the side-chain amide nitrogen in l-γ-glutamylamine substrates resulted in a dramatic decrease in substrate properties for gGACT thus providing an explanation of why gGACT does not act on l-γ-glutamyl amino acids except for l-γ-glutamylglycine. Placement of substituents on carbons further removed from the side-chain amide nitrogen in l-γ-glutamylamines restored activity for gGACT, and l-γ-glutamylneohexylamine 19 had a higher specificity constant (k _cat /K _m) than 1. gGACT did not exhibit any stereospecificity in the amide region of l-γ-glutamylamine substrates. In addition, analogues (26–30) with heteroatom substitutions for the γ methylene position of the l-γ-glutamyl moiety were examined. Several thiocarbamoyl derivatives of l-cysteine (28–30) were excellent substrates for gGACT.     

The Synthesis of l-dopa-l-Tyr and The Interaction of l-dopa-l-Tyr With ctDNA

l-dopa-l-Tyr was synthesized by Fmoc solid-phase peptide synthesis, purified by reversed-phase HPLC and characterized by using ¹H, ¹³C NMR and ESI–MS analyses. The interaction of l-dopa-l-Tyr and l-dopa with ctDNA has been investigated respectively by UV–vis absorption and fluorescence spectroscopy. The results showed that both l-dopa and l-dopa-l-Tyr interacted with ctDNA through intercalative mode and l-dopa-l-Tyr showed a higher affinity for DNA. Meanwhile, compared with the free l-dopa, gel electrophoresis assay also demonstrated that l-dopa-l-Tyr interacted with DNA by intercalation.     

Properties, metabolisms, and applications of l-proline analogues

Due to the unique role of l-proline in the folding and structure of protein, a variety of synthetic proline analogues have been developed. l-Proline analogues have been proven to be valuable reagents for studying cellular metabolism and the regulation of macromolecule synthesis in both prokaryotic and eukaryotic cells. In addition to these fundamental researches, they are useful compounds for industrial use. For instance, microorganisms that overproduce l-proline have been obtained by isolating mutants resistant to l-proline analogues. They are also promising candidates for tuning the biological, pharmaceutical, or physicochemical properties of naturally occurring or de novo designed peptides. Among l-proline analogues, l-azetidine-2-carboxylic acid (l-AZC) is a toxic non-proteinogenic amino acid originally found in lily of the valley plants and trans-4-hydroxy-l-proline (4-l-THOP) is the most abundant component of mammalian collagen. Many hydroxyprolines (HOPs), such as 4-l-THOP and cis-4-hydroxy-l-proline (4-l-CHOP), are useful chiral building blocks for the organic synthesis of pharmaceuticals. In addition, l-AZC and 4-l-CHOP, which are potent inhibitors of cell growth, have been tested for their antitumor activity in tissue culture and in vivo. In this review, we describe the recent discoveries regarding the physiological properties and microbial production and metabolism of l-proline analogues, particularly l-AZC and HOPs. Their applications in fundamental research and industrial use are also discussed.     

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.     

Orientation of the tryptophanyl side chain in [(d)TrpA1] and [TrpA1]insulin

Insulin analogues withl- andd-tryptophan instead of glycine in A1 permit an estimate of the proximity relationship between the indole residue of tryptophan and B19-tyrosine by evaluation of singlet-singlet resonance energy transfer. A significantly higher transfer efficiency is observed with [(d)Trp^A1]insulin than with the [Trp^A1]analogue. On the basis of this result it is possible to deduce the arrangement of the side chains and the α-amino groups in position A1 of [(d)Trp^A1] and [Trp^A1]insulin.     

l-Arginine and its metabolites in kidney and cardiovascular disease

l-Arginine is a semi essential amino acid synthesised from glutamine, glutamate and proline via the intestinal-renal axis in humans and most mammals. l-Arginine degradation occurs via multiple pathways initiated by arginase, nitric-oxide synthase, Arg: glycine amidinotransferase, and Arg decarboxylase. These pathways produce nitric oxide, polyamines, proline, glutamate, creatine and agmatine with each having enormous biological importance. Several disease are associated to an l-arginine impaired levels and/or to its metabolites: in particular various l-arginine metabolites may participate in pathogenesis of kidney and cardiovascular disease. l-Arginine and its metabolites may constitute both a marker of pathology progression both the rationale for manipulating l-arginine metabolism as a strategy to ameliorate these disease. A large number of studies have been performed in experimental models of kidney disease with sometimes conflicting results, which underlie the complexity of Arg metabolism and our incomplete knowledge of all the mechanisms involved. Moreover several lines of evidence demonstrate the role of l-arg metabolites in cardiovascular disease and that l-arg administration role in reversing endothelial dysfunction, which is the leading cause of cardiovascular diseases, such as hypertension and atherosclerosis. This review will discuss the implication of the mains l-arginine metabolites and l-arginine-derived guanidine compounds in kidney and cardiovascular disease considering the more recent literature in the field.     

Characterization of a recombinant l-rhamnose isomerase from Bacillus subtilis and its application on production of l-lyxose and l-mannose

The gene of an l-rhamnose isomerase (RhaA) from Bacillus subtilis was cloned to the pET28a(+) and then expressed in the E. coli ER2566. The expressed enzyme was purified with a specific activity of 3.58 U/mg by His-Trap affinity chromatography. The recombinant enzyme existed as a 194 kDa tetramer and the maximal activity was observed at pH 8.0 and 60°C. The RhaA displayed activity for l-rhamnose, l-lyxose, l-mannose, d-allose, d-gulose, d-ribose, and l-talose, among all aldopentoses and aldohexoses and it showed enzyme activity for l-form monosaccharides such as l-rhamnose, l-lyxose, l-mannose, and l-talose. The catalytic efficiency (k _cat/K _m) of the recombinant enzyme for l-rhamnose, l-lyxose, and l-mannose were 7,460, 1,013, and 258 M/sec. When l-xylulose 100 g/L and l-fructose 100 g/L were used as substrates, the optimum concentrations of RpiB were determined with 6 and 15 U/mL, respectively. The l-lyxose 40 g/L was produced from l-xylulose 100 g/L by the enzyme during 60 min, while l-mannose 25 g/L was produced from l-fructose 100 g/L for 80 min. The results suggest that RhaA from B. subtilis is a potential producer of l-form monosaccharides.     

Pressor response to l-cysteine injected into the cisterna magna of conscious rats involves recruitment of hypothalamic vasopressinergic neurons

The sulfur-containing non-essential amino acid l-cysteine injected into the cisterna magna of adult conscious rats produces an increase in blood pressure. The present study examined if the pressor response to l-cysteine is stereospecific and involves recruitment of hypothalamic vasopressinergic neurons and medullary noradrenergic A1 neurons. Intracisternally injected d-cysteine produced no cardiovascular changes, while l-cysteine produced hypertension and tachycardia in freely moving rats, indicating the stereospecific hemodynamic actions of l-cysteine via the brain. The double labeling immunohistochemistry combined with c-Fos detection as a marker of neuronal activation revealed significantly higher numbers of c-Fos-positive vasopressinergic neurons both in the supraoptic and paraventricular nuclei and tyrosine hydroxylase containing medullary A1 neurons, of l-cysteine-injected rats than those injected with d-cysteine as iso-osmotic control. The results indicate that the cardiovascular responses to intracisternal injection of l-cysteine in the conscious rat are stereospecific and include recruitment of hypothalamic vasopressinergic neurons both in the supraoptic and paraventricular nuclei, as well as of medullary A1 neurons. The findings may suggest a potential function of l-cysteine as an extracellular signal such as neuromodulators in central regulation of blood pressure.     

Synthesis of enantiomeric pureCis-dichloroplatinum(II) amino acid complexes

The reaction of potassium tetrachloroplatinate(II) with six representative sulfurcontaining amino acids, namely,d- andl-cysteine,d- andl-methionine and its methyl ester hydrochloride gives the corresponding enantiomerically purecis-dichloroplatinum(II) complexes. This represents the first reported series of well-characterized enantiomerically pure platinum(II) complexes for bothd- andl-amino acids. The spectroscopic properties, including IR,¹H-NMR, and¹³C NMR, of these complexes and their configuration are discussed.