Phenylalanine Oxidase Research Articles

Enzyme histochemical characterization of orbital glands in fetuses of Indian buffalo (Bubalus bubalis).

The orbital glands, viz. lacrimal gland, superficial and deep gland of third eyelid (LG, SGT and HG), are important for normal eye functions. These glands have different functions in various animals. The information about the enzyme histochemical nature of prenatal orbital glands in Indian buffalo seems to be unavailable. Therefore, the study was planned on orbital glands of six full term recently died fetuses from animals with dystocia. The frozen sections of all these glands were subjected to standard localization protocols for Alkaline Phosphatase (AKPase), Glucose 6 phosphatase (G-6-Pase), Lactate dehydrogenase (LDH), Succinate dehydrogenase (SDH), Glucose 6 phosphate dehydrogenase (G-6-PD), Nicotinamide Adenine Dinucleotide Hydrogen Diaphorase (NADHD), Nicotinamide Adenine Dinucleotide Phosphate Hydrogen diaphorase (NADPHD), Dihydroxy phenylalanine oxidase (DOPA-O), Tyrosinase, non-specific esterase (NSE) and Carbonic anhydrase (CAse). The results revealed a mixed spectrum of reaction for the above enzymes in LG, SGT and HG which ranged from moderate (for LDH in SGT) to intense (for most of the enzymes in all three glands). However, DOPA-O, Tyrosinase and CAse did not show any reaction. From the present study, it can be postulated that the orbital glands of fetus have a high activity of metabolism as it has many developmental and functional activities which were mediated with the higher activity of the enzymes involved.

Emerging Role of IL-4–Induced Gene 1 as a Prognostic Biomarker Affecting the Local T-Cell Response in Human Cutaneous Melanoma

Several studies have emphasized the importance of immune composition of the melanoma microenvironment for clinical outcome. The contribution of IL4I1, a phenylalanine oxidase with immunoregulatory functions, has not been yet explored. Here we studied a primary cutaneous melanoma series from stage I-III patients to investigate the association between in situ IL4I1 expression and clinical parameters or tumor-infiltrating T-cell subsets. IL4I1 was detected in 87% of tumors and was mainly expressed by tumor-associated macrophages and very rare FoxP3+ regulatory T cells. The proportion of IL4I1+ cells was higher in patients with an ulcerated melanoma or with a positive sentinel lymph node and tended to correlate with a rapid relapse and shorter overall survival. This proportion also correlated positively with the presence of regulatory T cells and negatively with the presence of cytotoxic CD8+ T cells. The location of IL4I1+ cells may also be relevant to predict prognosis, because their presence near tumor cells was associated with sentinel lymph node invasion and higher melanoma stage. Collectively, our data show that IL4I1+ cells shape the T-cell compartment and are associated with a higher risk of poor outcome in melanoma, supporting a key role for IL4I1 in immune evasion.

An Overview of l-Amino Acid Oxidase Functions from Bacteria to Mammals: Focus on the Immunoregulatory Phenylalanine Oxidase IL4I1.

l-amino acid oxidases are flavin adenine dinucleotide-dependent enzymes present in all major kingdom of life, from bacteria to mammals. They participate in defense mechanisms by limiting the growth of most bacteria and parasites. A few mammalian LAAOs have been described, of which the enzyme “interleukin-4 induced gene 1” (IL4I1) is the best characterized. IL4I1 mainly oxidizes l-phenylalanine. It is a secreted enzyme physiologically produced by antigen presenting cells of the myeloid and B cell lineages and T helper type (Th) 17 cells. Important roles of IL4I1 in the fine control of the adaptive immune response in mice and humans have emerged during the last few years. Indeed, IL4I1 inhibits T cell proliferation and cytokine production and facilitates naïve CD4+ T-cell differentiation into regulatory T cells in vitro by limiting the capacity of T lymphocytes to respond to clonal receptor stimulation. It may also play a role in controlling the germinal center reaction for antibody production and limiting Th1 and Th17 responses. IL4I1 is expressed in tumor-associated macrophages of most human cancers and in some tumor cell types. Such expression, associated with its capacity to facilitate tumor growth by inhibiting the anti-tumor T-cell response, makes IL4I1 a new potential druggable target in the field of immunomodulation in cancer.

Characterization of a novel l -phenylalanine oxidase from Coprinopsis cinereus and its application for enzymatic production of phenylpyruvic acid

Abstract A novel l -phenylalanine oxidase gene from a species of mushroom Coprinopsis cinereus was cloned. With l -amino acid oxidase from Hebeloma cylindrosporum, which is the closest one, it shared only 30.6% sequence identity. This recombinant protein was expressed in Escherichia coli, purified and biochemically characterized. It contained 778 amino acids and was quite different compared with all previously studied enzymes. This enzyme exhibited highest specific activity of 6.04 U/mg towards l -phenylalanine and the optimal pH, temperature of the enzyme catalyzed reaction were 8.5 and 45 °C. The enzyme was stable up to 55 °C within pH range 7.0–9.5. It could oxidize l -phenylalanine to phenylpyruvic acid at high titer (8.1 ± 0.1 g/L), conversion ratio (97.4 ± 0.2%) and productivity (1.02 ± 0.01 g/L h) within 8 h. More importantly, it specifically catalyzed the oxidation of l -phenylalanine with racemic null , l -phenylalanine mixture as substrate. In general, these properties rendered it a useful catalyst in several industrial manufacturers.

Interleukin-4 induced 1 (IL4I1) promotes central nervous system remyelination.

This scientific commentary refers to ‘IL4I1 augments CNS remyelination and axonal protection by modulating T cell driven inflammation’, by Psachoulia et al. (doi:10.1093/brain/aww254) . Multiple sclerosis is an inflammatory demyelinating disease of the CNS characterized by infiltration of immune cells and progressive damage to myelin sheaths and axons (Lorscheider et al. , 2016). Most patients initially develop relapsing-remitting disease (RRMS), in which acute neurological deficits are interspersed with periods of partial or complete recovery. However, up to 65% of patients with RRMS eventually develop secondary progressive multiple sclerosis (SPMS), in which disability increases continually. Conversion to SPMS is associated with a relatively poor prognosis, in part due to the limited effectiveness of disease-modifying therapies in the progressive phase (Lorscheider et al. , 2016). This in turn partly reflects the complex interactions between the multiple pathophysiological mechanisms responsible for demyelination and neurodegeneration. However, there is also compelling evidence that a decline in regenerative capacity occurs in the brain in chronic multiple sclerosis, possibly due to dysregulation of inflammation (Fitzner and Simons, 2010). Unravelling the mechanisms that link the immune system with CNS damage and repair therefore represents both a challenge and an opportunity for multiple sclerosis research. In this issue of Brain , Psachoulia et al. (2016) exploit a published remyelination transcriptome obtained from laser capture microdissected CNS lesions induced in the rodent CNS by lysolecithin/lysophosphatidylcholine (LPC) (Huang et al. , 2011), to identify novel markers of remyelination. Of the more than 8000 differentially regulated genes, Il4i1 , a phenylalanine oxidase secreted mainly by myeloid antigen presenting cells (APCs), was among the most strongly upregulated (4-fold increase at 14 days versus 5 days post-lesion). In situ hybridization demonstrated that Il4i1 was predominantly expressed in cells with morphology reminiscent of foamy macrophages, beginning as early as 10–14 days post-lesion, a …

Alterations of the immunosuppressive IL4I1 enzyme activity induced by naturally occurring SNP/mutations

The immunosuppressive phenylalanine oxidase interleukin 4-induced gene 1 (IL4I1), primarily produced by antigen-presenting cells, inhibits T-cell proliferation and promotes the generation of Foxp3(+) regulatory T cells in vitro. Highly expressed by tumour-associated macrophages from human cancers, IL4I1 has a potential role in immune evasion from the anti-tumour immune response. We have reviewed single-nucleotide polymorphisms (SNPs) and mutations described for the exon 4 of the IL4I1 isoform 1, which is expressed in lymphoid tissue. Two of them were expressed in an exogenous system to analyse their effect on the enzymatic activity. The N92D SNP leads to a hyperactive enzyme, while the R102G mutation is hypomorphic. Moreover, we show that IL4I1 activity is not only directed against phenylalanine, as initially described, but also at a lower level against arginine. These data pave the way to more extensive analyses of the mutational state of IL4I1 in pathological conditions such as cancer, where its participation in immune system dysfunctions may have therapeutic implications.

The immunosuppressive enzyme IL4I1 promotes FoxP3(+) regulatory T lymphocyte differentiation.

IL4I1 (interleukin-4-induced gene 1) is a phenylalanine oxidase produced mainly by APCs of myeloid origin, and converts phenylalanine (Phe) to phenylpyruvate, hydrogen peroxide, and ammonia. We have previously shown that IL4I1 is highly expressed by tumor-associated macrophages from various human cancers and facilitates immune evasion from the cytotoxic response in a murine tumor model. Indeed, IL4I1 inhibits T-cell proliferation via hydrogen peroxide toxicity on effector/memory Tcells. Here, we explored the effect of IL4I1 on naïve CD4(+) T-cell differentiation. We show that IL4I1 stimulates the generation of Foxp3(+) regulatory T (Treg) cells in vitro from human and mouse Tcells. This effect was observed with IL4I1 from different sources, including the naturally produced enzyme. Conversely, IL4I1 limits Th1 and Th2 polarization while modifying the Th17 phenotype, in particular, by inducing its own production. Analysis of Treg-cell induction under conditions of Phe deprivation and hydrogen peroxide addition suggests that Phe consumption by the enzyme participates in Treg-cell enrichment. In line with this hypothesis, IL4I1 inhibits mTORC1 signaling shortly after T-cell activation. Thus, the IL4I1 enzyme may act on Tcells both by direct inhibition of effector cell proliferation and by indirect immunoregulation mediated by Treg-cell induction.

Catalyzed Determination of Phenylalanine with a Mimic Enzyme Constructed of β-Cyclodextrain Derivant

Phenylalanine was catalyzed and determined with mimic phenylalanine oxidase constructed by the reaction of β-cyclodextrin (or β-CD) and m-carboxyl benzenesulfonyl chloride with iron trichloride in hydrogen peroxide. The method is simple and convenient. The linear range of this method is 0–∼0.8233 mmol/L, the detection limit is 8.354 μmol/L, the RSD is 0.96% (n = 8), and the recovery rate is 95.8–∼103.5% when used to test an edulcorator (aspartame) sample.

Bovine pancreatic elastase II cleaves Gln-Ile bond.

A peptidase (GICP) that cleaves the Gln-Ile bond of a peptide Gly-Ile-Asp-Val-Gln-Ile-Tyr(T-1), a sequence in phenylalanine oxidase, was purified from bovine pancreas. The purified enzyme had an Mr of approximately 29,000, as determined by SDS-PAGE, and its N-terminal sequence was identical to that of bovine pancreatic elastase II. The enzyme released Gly-Ile-Asp-Val-Gln and Ile-Tyr from T-1 (Km = 8.3 microM k(cat) = 2.1 s(-1)) and the catalytic efficiency (2.6 X 10(5) M(-1)s(-1)) was comparable to those of elastase II from porcine pancreas and rat mesenteric arterial bed perfusate. The P1 site specificity of GICP toward oxidized insulin A and B chains suggested that major cleavage sites were the peptide bond at the C-terminal side of Gln, Leu, His, and Tyr residues.

Isolation, identification and characterisation of a soil bacterium producing an enzyme with l-phenylalanine oxidase activity

A gram-positive, mesophilic bacterium which assimilated l-phenylalanine but which failed to utilise l-tyrosine was isolated from soil. The isolate, identified as a strain of Bacillus carotarum, converted l-phenylalanine to phenylpyruvate with the initial step catalysed by an inducible, intracellular enzyme which possessed l-phenylalanine oxidase activity. Phenylalanine oxidase has not been previously reported in Gram-positive bacteria, although there are a few examples of non-specific l-amino acid oxidases with activity towards l-phenylalanine. The isolate grew abundantly on complex media but failed to synthesise significant amounts of the enzyme in the absence of l-phenylalanine. The highest enzyme levels were achieved in a chemically defined minimal salts medium containing the amino acid at 10 g/l as the primary carbon and energy source.

Regulation of phenylalanine oxidase synthesis in Proteus mirabilis

Cells of Proteus mirabilis could oxidize L-phenylalanine to phenylpyruvate only when grown in the presence of a number of amino acids, particularly, L-alanine, L-asparagine, L-glutamate, and L-glutamine. Production of phenylalanine oxidase was slowly lost upon growth in a minimal medium containing ammonium ions as a nitrogen source but was reversed by the addition of casein hydrolysate. Oxidase activity as well as a phenylalanine-dichlorophenolindophenol (DCIP) reductase activity increased in P. mirabilis only during cell multiplication. Both rifampin and nalidixic acid caused inhibition of oxidase synthesis. A phenylalanine-active transport was found to be operative when bacteria were grown in the absence of added amino acids. After anaerobic growth, cells of P. mirabilis had lost their ability to carry the phenylalanine oxidase reaction when assayed in the presence of air, and nitrate could not be used as an electron acceptor for the oxidation of phenylalanine. However, some phenylalanine-dichlorophenolindophenol reductase activity was still present in anaerobic bacteria at the early stage of cell multiplication.

Urinary metabolites of aromatic amino acids in schizophrenia

Tf~RS have been a nimber ~ reports that the urine of schisophx~enics contains unusual aromatio com~peunda not Pound in normal urine, nor in that from other psychotica . i-5 Moreover, one group ad waa~kers~ have offered evidence that these unusual metabolites are derived from dietary pher~rlalanine and tyrosine and can be eliminated from schizophrenics urine by feeding a diet low in these two amino acids. This evidence can be ezplaiaed if there is an impairnient in schizophrenics of the phenTplalanine aQidase system. In other work6 ' 7 evidence has been pa~esented for an excessive breakdam of ascorbic acid in schizophrenics . Nos ascorbic acid is an activator of orge enzyme of the phenylalanine oxidase system, ~-hydro~pher~ylpyruvate aad.daae .8 Consequently, the obsertrations of the paevious worlrors could be ezplained by a partial inhibition of this enzyme in the tissues aâ schizophrenics . To examine this hypothesis we have compared the ara®atic metabolites gresent in schizophrenics' urine with those in urine from a na~rmal group and from a group of non-schizophrenic psychotics . We have looked particularly for the paesenee and absence of the taro compounds knosn to be excreted. during the inhibition of ~-hydroxypher~ylpyruvate ox;.dase : and ~-hydrozyphenyllactate. 8