4-Hydroxyphenylpyruvic acid

Hydroxy(phenyl)pyruvic acid reductase in Actaea racemosa L.: a putative enzyme in cimicifugic and fukinolic acid biosynthesis.[Pubmed:38549005]

Planta. 2024 Mar 28;259(5):102.

Hydroxy(phenyl)pyruvic acid reductase from Actaea racemosa catalyzes dual reactions in reducing 4-Hydroxyphenylpyruvic acid as well as beta-hydroxypyruvic acid. It thus qualifies to be part of fukinolic and cimicifugic acid biosynthesis and also photorespiration. The accumulation of fukinolic acid and cimicifugic acids is mainly restricted to Actaea racemosa (Ranunculaceae) and other species of the genus Actaea/Cimicifuga. Cimicifugic and fukinolic acids are composed of a hydroxycinnamic acid part esterified with a benzyltartaric acid moiety. The biosynthesis of the latter is unclear. We isolated cDNA encoding a hydroxy(phenyl)pyruvic acid reductase (GenBank OR393286) from suspension-cultured material of A. racemosa (ArH(P)PR) and expressed it in E. coli for protein production. The heterologously synthesized enzyme had a mass of 36.51 kDa and catalyzed the NAD(P)H-dependent reduction of 4-Hydroxyphenylpyruvic acid to 4-hydroxyphenyllactic acid or beta-hydroxypyruvic acid to glyceric acid, respectively. The optimal temperature was at 38 degrees C and the pH optimum at pH 7.5. NADPH is the preferred cosubstrate (K(m) 23 +/- 4 microM). Several substrates are accepted by ArH(P)PR with beta-hydroxypyruvic acid (K(m) 0.26 +/- 0.12 mM) followed by 4-Hydroxyphenylpyruvic acid (K(m) 1.13 +/- 0.12 mM) as the best ones. Thus, ArH(P)PR has properties of beta-hydroxypyruvic acid reductase (involved in photorespiration) as well as hydroxyphenylpyruvic acid reductase (possibly involved in benzyltartaric acid formation).

Dysbiosis of gut microbiota and metabolites during AIDS: implications for CD4 + T cell reduction and immune activation.[Pubmed:38061029]

AIDS. 2024 Apr 1;38(5):633-644.

OBJECTIVE: Identifying the gut microbiota associated with host immunity in the AIDS stage. DESIGN: We performed a cross-sectional study. METHODS: We recruited people with HIV (PWH) in the AIDS or non-AIDS stage and evaluated their gut microbiota and metabolites by using 16S ribosomal RNA (rRNA) sequencing and liquid chromatography-mass spectrometry (LC-MS). Machine learning models were used to analyze the correlations between key bacteria and CD4 + T cell count, CD4 + T cell activation, bacterial translocation, gut metabolites, and KEGG functional pathways. RESULTS: We recruited 114 PWH in the AIDS stage and 203 PWH in the non-AIDS stage. The alpha-diversity of gut microbiota was downregulated in the AIDS stage ( P < 0.05). Several machine learning models could be used to identify key gut microbiota associated with AIDS, including the logistic regression model with area under the curve (AUC), sensitivity, specificity, and Brier scores of 0.854, 0.813, 0.813, and 0.160, respectively. The decreased key bacteria ASV1 ( Bacteroides sp.), ASV8 ( Fusobacterium sp.), ASV30 ( Roseburia sp.), ASV37 ( Bacteroides sp.), and ASV41 ( Lactobacillus sp.) in the AIDS stage were positively correlated with the CD4 + T cell count, the EndoCAb IgM level, 4-Hydroxyphenylpyruvic acid abundance, and the predicted cell growth pathway, and negatively correlated with the CD3 + CD4 + CD38 + HLA-DR + T cell count and the sCD14 level. CONCLUSION: Machine learning has the potential to recognize key gut microbiota related to AIDS. The key five bacteria in the AIDS stage and their metabolites might be related to CD4 + T cell reduction and immune activation.

Expression Patterns and Functional Analysis of Three SmTAT Genes Encoding Tyrosine Aminotransferases in Salvia miltiorrhiza.[Pubmed:37958559]

Int J Mol Sci. 2023 Oct 25;24(21):15575.

Tyrosine aminotransferase (TAT, E.C. 2.6.1.5) is a pyridoxal phosphate-dependent aminotransferase that is widely found in living organisms. It catalyzes the transfer of the amino group on tyrosine to alpha-ketoglutarate to produce 4-Hydroxyphenylpyruvic acid (4-HPP) and is the first enzyme for tyrosine degradation. Three SmTATs have been identified in the genome of Salvia miltiorrhiza (a model medicinal plant), but their information is very limited. Here, the expression profiles of the three SmTAT genes (SmTAT1, SmTAT2, and SmTAT3) were studied. All three genes expressed in different tissues and responded to methyl jasmonate stimuli. SmTAT proteins are localized in the cytoplasm. The recombinant SmTATs were subjected to in vitro biochemical properties. All three recombinant enzymes had TAT activities and SmTAT1 had the highest catalytic activity for tyrosine, followed by SmTAT3. Also, SmTAT1 preferred the direction of tyrosine deamination to 4-HPP, while SmTAT2 preferred transamination of 4-HPP to tyrosine. In parallel, transient overexpression of SmTATs in tobacco leaves revealed that all three SmTAT proteins catalyzed tyrosine to 4-HPP in vivo, with SmTAT1 exhibiting the highest enzymatic activity. Overall, our results lay a foundation for the production of tyrosine-derived secondary metabolites via metabolic engineering or synthetic biology in the future.

Identification of Serum Biomarkers of Ischemic Stroke in a Hypertensive Population Based on Metabolomics and Lipidomics.[Pubmed:37806545]

Neuroscience. 2023 Nov 21;533:22-35.

Hypertensive individuals are at a high risk of stroke, and thus, prevention of stroke in hypertensive patients is essential. Metabolomics and lipidomics can be used to identify diagnostic biomarkers and conduct early assessments of stroke risk in hypertensive populations. In this study, serum samples were collected from 30 hypertensive ischemic stroke (IS), 30 matched hypertensive and 30 matched healthy participants. Metabolomics and lipidomics analyses were conducted via liquid chromatography-tandem mass spectrometry, and the data were analyzed using multivariate and univariate statistical methods. A random forest algorithm and binary logistic regression were used to screen the biomarkers and establish diagnostic model. We detected 21 differential metabolites and 38 differential lipids between the hypertensive IS and healthy group. Moreover, we found 18 differential metabolites and 31 differential lipids between the hypertensive IS and hypertension group. In particular, the following seven metabolites or lipids distinguished the hypertensive IS from the healthy group: 4-Hydroxyphenylpyruvic acid, cafestol, phosphatidylethanolamine (PE) (18:0p/18:2), PE (16:0e/20:4), (O-acyI)-1-hydroxy fatty acid (36:3), PE (16:0p/20:3) and PE (18:1p/18:2) (rep). The following seven biomarkers distinguished the hypertensive IS from the hypertension group: diglyceride (DG) (20:1/18:2), PE (18:0p/18:2), PE (16:0e/22:5), phosphatidylcholine (40:7), dimethylphosphatidylethanolamine (50:3), DG (18:1/18:2), and 4-Hydroxyphenylpyruvic acid. The aforementioned panels had good diagnostic and predictive ability for hypertensive IS. Our study determines the metabolomic and lipidomic profiles of hypertensive IS patients and thereby identifies potential biomarkers of the presence of IS in hypertensive populations.

Comparative analysis of three SmHPPR genes encoding hydroxyphenylpyruvate reductases in Salvia miltiorrhiza.[Pubmed:37797780]

Gene. 2024 Jan 20;892:147868.

Hydroxyphenylpyruvate reductase (HPPR) is an enzyme that is involved in the biosynthesis of hydrophilic phenolic acids in Salvia miltiorrhiza, which is a model medicinal plant. Three SmHPPR genes have been identified in the S. miltiorrhiza genome; however, only one has been functionally analyzed. Here, we cloned three SmHPPR genes (SmHPPR1, SmHPPR2, and SmHPPR3) from the cDNA of S. miltiorrhiza, and their expression profiles were studied. The expression levels of SmHPPR1 were significantly higher than those of SmHPPR2 and SmHPPR3, where SmHPPR1 revealed the highest level in stems, while SmHPPR2 and SmHPPR3 exhibited the highest level in flowers. SmHPPR1, SmHPPR2, and SmHPPR3 are localized in the cytoplasm. All three recombinant enzymes had HPPR activities and catalyzed the reduction of 4-Hydroxyphenylpyruvic acid (pHPP) to 4-hydroxyphenyllactic acid (pHPL), with SmHPPR1 showing the highest activity. The transient over-expression of SmHPPR1, SmHPPR2, and SmHPPR3 in the leaves of Nicotiana benthamiana promoted the production of pHPL, which indicated that all three SmHPPRs had in vivo activities. Overall, between the three homologs, SmHPPR1 plays a dominant role in catalyzing pHPP to pHPL, which provides new insights into the biosynthesis of phenolic acids in S. miltiorrhiza.

Targeted metabolomics analysis for serum Helicobacter pylori-positive based on liquid chromatography-tandem mass spectrometry.[Pubmed:36898359]

Biomed Chromatogr. 2023 Jun;37(6):e5622.

Helicobacter pylori (H. pylori), as a harmful bacteria associated with gastric cancer, can have adverse effects on human normal flora and metabolism. However, the effects of H. pylori on human metabolism have not been fully elucidated. The (13) C breathing test was used as the basis for distinguishing negative and positive groups. Serum samples were collected from the two groups for targeted quantitative metabolomics detection; multidimensional statistics were used, including partial least squares discriminant analysis (PLS-DA), principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA), and differential metabolites were screened. Unidimensional statistics combined with multidimensional statistics were used to further screen potential biomarkers, and finally pathway analysis was performed. SPSS 21.0 software package was used for statistical analysis of experimental data. Multivariate statistical analysis such as PLS-DA, PCA, and OPLS-DA was performed using Simca-P 13.0 to search for differential metabolites. This study confirmed that H. pylori caused significant changes in human metabolism. In this experiment, 211 metabolites were detected in the serum of the two groups. Multivariate statistical analysis showed that PCA of metabolites was not significantly different between the two groups. PLS-DA indicated that the serum of the two groups was well clustered. There were significant differences in metabolites between OPLS-DA groups. By setting the variable importance in projection (VIP) threshold as one and the corresponding P-value <0.05, a total of 40 metabolites were screened in this study. P <0.05 and mid R:log2FCmid R:>0 (where FC is the fold change) were used together as a unidimensional statistical filter condition. The analysis found that the expression of 15 metabolites such as propionic acid, acetic acid, adipic acid increased, and the metabolism of six products such as deoxycholic acid (DCA), 4-Hydroxyphenylpyruvic acid, pyruvic acid decreased. P <0.05, false discovery rate <0.5, mid R:log2FCmid R:>1, and OPLSDA_VIP>1 were used together as a condition for filter screening potential biomarkers. Four potential biomarkers were screened, which were sebacic acid, isovaleric acid, DCA, and indole-3-carboxylic acid. Finally, the different metabolites were added to the pathway-associated metabolite sets (SMPDB) library for the corresponding pathway enrichment analysis. The significant abnormal metabolic pathways were taurine and subtaurine metabolism, tyrosine metabolism, glycolysis or gluconeogenesis, pyruvate metabolism, etc. This study shows that H. pylori has an impact on human metabolism. Not only a variety of metabolites have significant changes, but also metabolic pathways are abnormal, which may be the reason for the high risk of H. pylori causing gastric cancer.

Comprehensive Biotransformation Analysis of Phenylalanine-Tyrosine Metabolism Reveals Alternative Routes of Metabolite Clearance in Nitisinone-Treated Alkaptonuria.[Pubmed:36295829]

Metabolites. 2022 Sep 29;12(10):927.

Metabolomic analyses in alkaptonuria (AKU) have recently revealed alternative pathways in phenylalanine-tyrosine (phe-tyr) metabolism from biotransformation of homogentisic acid (HGA), the active molecule in this disease. The aim of this research was to study the phe-tyr metabolic pathway and whether the metabolites upstream of HGA, increased in nitisinone-treated patients, also undergo phase 1 and 2 biotransformation reactions. Metabolomic analyses were performed on serum and urine from patients partaking in the SONIA 2 phase 3 international randomised-controlled trial of nitisinone in AKU (EudraCT no. 2013-001633-41). Serum and urine samples were taken from the same patients at baseline (pre-nitisinone) then at 24 and 48 months on nitisinone treatment (patients N = 47 serum; 53 urine) or no treatment (patients N = 45 serum; 50 urine). Targeted feature extraction was performed to specifically mine data for the entire complement of theoretically predicted phase 1 and 2 biotransformation products derived from phenylalanine, tyrosine, 4-Hydroxyphenylpyruvic acid and 4-hydroxyphenyllactic acid, in addition to phenylalanine-derived metabolites with known increases in phenylketonuria. In total, we observed 13 phase 1 and 2 biotransformation products from phenylalanine through to HGA. Each of these products were observed in urine and two were detected in serum. The derivatives of the metabolites upstream of HGA were markedly increased in urine of nitisinone-treated patients (fold change 1.2-16.2) and increases in 12 of these compounds were directly proportional to the degree of nitisinone-induced hypertyrosinaemia (correlation coefficient with serum tyrosine = 0.2-0.7). Increases in the urinary phenylalanine metabolites were also observed across consecutive visits in the treated group. Nitisinone treatment results in marked increases in a wider network of phe-tyr metabolites than shown before. This network comprises alternative biotransformation products from the major metabolites of this pathway, produced by reactions including hydration (phase 1) and bioconjugation (phase 2) of acetyl, methyl, acetylcysteine, glucuronide, glycine and sulfate groups. We propose that these alternative routes of phe-tyr metabolism, predominantly in urine, minimise tyrosinaemia as well as phenylalanaemia.

Insights into growth-promoting effect of nanomaterials: Using transcriptomics and metabolomics to reveal the molecular mechanisms of MWCNTs in enhancing hyperaccumulator under heavy metal(loid)s stress.[Pubmed:35882170]

J Hazard Mater. 2022 Oct 5;439:129640.

Carbon nanotubes present potential applications in soil remediation, particularly in phytoremediation. Yet, how multi-walled carbon nanotubes (MWCNTs) induced hyperaccumulator growth at molecular level remains unclear. Here, physio-biochemical, transcriptomic, and metabolomic analyses were performed to determine the effect of MWCNTs on Solanum nigrum L. (S. nigrum) growth under cadmium and arsenic stresses. 500 mg/kg MWCNTs application significantly promoted S. nigrum growth, especially for root tissues. Specially, MWCNTs application yields 1.38-fold, 1.56-fold, and 1.37-fold enhancement in the shoot length, root length, and fresh biomass, respectively. Furthermore, MWCNTs significantly strengthened P and Fe absorption in roots, as well as the activities of antioxidative enzymes. Importantly, the transcriptomic analysis indicated that S. nigrum gene expression was sensitive to MWCNTs, and MWCNTs upregulated advantageous biological processes under heavy metal(loid)s stress. Besides, MWCNTs reprogramed metabolism that related to defense system, leading to accumulation of 4-Hydroxyphenylpyruvic acid (amino acid), 4-hydroxycinnamic acid (xenobiotic), and (S)-abscisic acid (lipid). In addition, key common pathways of differentially expressed metabolites and genes, including "tyrosine metabolism" and "isoquinoline alkaloid biosynthesis" were selected via integrating transcriptome and metabolome analyses. Combined omics technologies, our findings provide molecular mechanisms of MWCNTs in promoting S. nigrum growth, and highlight potential application of MWCNTs in soil remediation.

Method development and validation for analysis of phenylalanine, 4-hydroxyphenyllactic acid and 4-hydroxyphenylpyruvic acid in serum and urine.[Pubmed:35822095]

JIMD Rep. 2022 Apr 3;63(4):341-350.

Alkaptonuria (AKU) is a rare debilitating autosomal recessive disorder of tyrosine (TYR) metabolism which results in a deficiency of the enzyme homogentisate 1,2-dioxygenase activity. Several studies have reported the metabolic changes in homogentisic acid (HGA) concentrations and subsequent deposition of an ochronotic pigment in connective tissues, especially cartilage. Treatment with nitisinone (NTBC) reduces urinary and circulating HGA, but its mode of action results in hypertyrosinaemia. The effect of NTBC on other metabolites in the TYR pathway has not been reported. Modification of the current reverse phase liquid chromatography tandem mass spectrometry methods for serum and urine to include phenylalanine (PHE), hydroxyphenyllactate (HPLA) and hydroxyphenylpyruvate (HPPA) has been validated. HPPA and HPLA (negative ionisation) eluted at 2.8 and 2.9 min respectively on an Atlantis C18 column with PHE (positive ionisation) eluting earlier at 2.4 min. Intra- and inter-assay accuracy was between 96.3% and 100.3% for PHE; 96.6% and 110.5% for HPLA and 95.0% and 107.8% for HPPA in both urine and serum. Precision, both inter- and intra-assay, was <10% for all analytes in both serum and urine. No significant issues with carry-over, stability or matrix interferences were seen in either the urine or serum assays. Measurement of serum and urine from AKU patients has demonstrated a robust, fully validated assay, appropriate for monitoring of patients with AKU and for demonstrating metabolite changes, following NTBC therapy.

The hydroperoxyl antiradical activity of natural hydroxycinnamic acid derivatives in physiological environments: the effects of pH values on rate constants.[Pubmed:35702430]

RSC Adv. 2022 May 18;12(24):15115-15122.

Hydroxycinnamic acid derivatives (HCA) are a type of phenolic acid that occurs naturally. HCA are widely known for their anti-inflammatory, anti-cancer, and especially antioxidant capabilities; however, a comprehensive study of the mechanism and kinetics of the antiradical activity of these compounds has not been performed. Here, we report a study on the mechanisms and kinetics of hydroperoxyl radical scavenging activity of HCA by density functional theory (DFT) calculations. The ability of HCA to scavenge hydroperoxyl radicals in physiological environments was studied. The results showed that HCA had moderate and weak HOO antiradical activity in pentyl ethanoate solvent, with the overall rate constant k (overall) = 8.60 x 10(1) - 3.40 x 10(4) M(-1) s(-1). The formal hydrogen transfer mechanism of phenyl hydroxyl groups defined this action. However, in water at physiological pH, 2-coumaric acid (1), 4-coumaric acid (2), caffeic acid (3), ferulic acid (4), sinapic acid (5) and 4-Hydroxyphenylpyruvic acid (7) exhibit a significant HOO antiradical activity with k (total) = 10(5) - 10(8) M(-1) s(-1) by the electron transfer reaction of the phenolate anions. Following a rise in pH levels in most of the studied substances, the overall rate constant varied. The acid 5 exhibited the highest HOO radical scavenging activity (log(k (overall)) = 4.6-5.1) at pH < 5; however, at pH = 5.4-8.8, the highest HOO radical scavenging activity were observed for 3 with log(k (overall)) = 5.2-5.7. At pH > 6.2, acids 2, 3, 4, and 5 presented the largest radical scavenging activity. By contrast, acid 3-coumaric acid (8) had the lowest antiradical activity at most pH values. Thus, the hydroperoxyl radical scavenging activity in pentyl ethanoate follows the order 3 > 5 > 1 approximately 2 approximately 4 approximately 6 (homovanillic acid) approximately 7 > 8, whereas it follows the order 3 > 2 approximately 4 approximately 5 > 6 approximately 7 > 1 > 8 in water at pH = 7.40. The activity of 1, 2, 3, 4, 5, 6, and 7 are faster than those of the reference Trolox, suggesting that these HCA could be useful natural antioxidants in the aqueous physiological environment.

[Shenling Baizhu Powder alleviates chronic inflammation to prevent type 2 diabetes of ZDF rats via intestinal flora].[Pubmed:35285199]

Zhongguo Zhong Yao Za Zhi. 2022 Feb;47(4):988-1000.

This study explored the mechanism of Shenling Baizhu Powder(SLBZP) in the prevention and treatment of type 2 diabetes from the perspective of flora disorder and chronic inflammation. Fifty rats were randomly divided into normal control group, model control group, low-dose SLBZP group, medium-dose SLBZP group, and high-dose SLBZP group, with 10 rats in each group. The rats of 5 weeks old were administrated by gavage with ultrapure water and different doses of SLBZP decoction. The basic indicators such as body weight and blood glucose were monitored every week, and stool and intestinal contents were collected from the rats of 9 weeks old for 16 S rRNA sequencing and metabolomic analysis. An automatic biochemical analyzer was used to measure the serum biochemical indicators, ELISA to measure serum insulin, and chipsets to measure leptin and inflammatory cytokines. The results showed that SLBZP reduced the body weight as well as blood glucose, glycosylated hemoglobin, and lipid levels. In the rats of 9 weeks, the relative abundance of Anaerostipes, Turicibacter, Bilophila, Ochrobactrum, Acinetobacter, and Prevotella decreased significantly in the model control group, which can be increased in the high-dose SLBZP group; the relative abundance of Psychrobacter, Lactobacillus, Roseburia and Staphylococcus significantly increased in the model control group, which can be down-regulated in the high-dose SLBZP group. The differential metabolites of intestinal flora included 4-Hydroxyphenylpyruvic acid, phenylpyruvic acid, octanoic acid, 3-indolepropionic acid, oxoglutaric acid, malonic acid, 3-methyl-2-oxovaleric acid, and methylmalonic acid. Moreover, SLBZP significantly lowered the levels of free insulin, insulin resistance and leptin resistance in rats. The variations in the serum levels of interleukin 1beta(IL-1beta) and monocyte chemoattractant protein-1(MCP-1) showed that SLBZP could alleviate chronic inflammation in rats. In conclusion, SLBZP can regulate intestinal flora and metabolites and relieve chronic inflammation to control obesity and prevent type 2 diabetes.

Formation of Coelenteramine from 2-Peroxycoelenterazine in the Ca(2+) -Binding Photoprotein Aequorin.[Pubmed:34971002]

Photochem Photobiol. 2022 Sep;98(5):1068-1076.

Aequorin consists of apoprotein (apoAequorin) and (S)-2-peroxycoelenterazine (CTZ-OOH) and is considered to be a transient-state complex of an enzyme (apoAequorin) and a substrate (coelenterazine and molecular oxygen) in the enzymatic reaction. The degradation process of CTZ-OOH in aequorin was characterized under various conditions of protein denaturation. By acid treatment, the major product from CTZ-OOH was coelenteramine (CTM), but not coelenteramide (CTMD), and no significant luminescence was observed. The counterparts of CTM from CTZ-OOH were identified as 4-Hydroxyphenylpyruvic acid (4HPPA) and 4-hydroxyphenylacetic acid (4HPAA) by liquid chromatography/electrospray ionization-time-of-flight mass spectrometry (LC/ESI-TOF-MS). In the luminescence reaction of aequorin with Ca(2+) , similar amounts of 4HPPA and 4HPAA were detected, indicating that CTM is formed by two pathways from CTZ-OOH through dioxetanone anion and not by hydrolysis from CTMD.