12-hydroxydodecanoic Acid Research Articles

Structure modeling-based characterization of ChnD, the 6-hydroxyhexanoate dehydrogenase from Acinetobacter sp. strain NCIMB 9871

α,ω-Dicarboxylic acids, ω-aminoalkanoic acids, and α,ω-diaminoalkanes are valuable building blocks for the production of biopolyesters and biopolyamides. One of the key steps in producing these chemicals is the oxidation of ω-hydroxycarboxylic acids using alcohol dehydrogenases (e.g., ChnD of Acinetobacter sp. NCIMB 9871). However, the reaction and structural features of these enzymes remain mostly undiscovered. Thereby, we have investigated characteristics of ChnD based on enzyme kinetics, substrate-docking simulations, and mutation studies. Kinetic analysis revealed a distinct preference of ChnD for medium chain ω-hydroxycarboxylic acids, with the highest catalytic efficiency of 18.0 mM−1s−1 for 12-hydroxydodecanoic acid among C6 to C12 ω-hydroxycarboxylic acids. The high catalytic efficiency was attributed to the positive interactions between the carboxyl group of the substrates and the guanidino group of two arginine residues (i.e., Arg62 and Arg266) in the substrate binding site. The ChnD_R62L variant showed the increased efficiency and affinity, particularly for fatty alcohols (i.e., C6–C10) and branched-chain fatty alcohols, such as 3-methyl-2-buten-1-ol. Overall, this study contributes to the deeper understanding of medium-chain primary aliphatic alcohol dehydrogenases and their applications for the production of industrially relevant chemicals such as α,ω-dicarboxylic acids, ω-aminoalkanoic acids, and α,ω-diaminoalkanes from renewable biomass.

Salivary metabolites are promising noninvasive biomarkers of drug-induced liver injury.

Drug-induced liver injury (DILI) is one of the most common adverse events of medication use, and its incidence is increasing. However, early detection of DILI is a crucial challenge due to a lack of biomarkers and noninvasive tests. To identify salivary metabolic biomarkers of DILI for the future development of noninvasive diagnostic tools. Saliva samples from 31 DILI patients and 35 healthy controls (HCs) were subjected to untargeted metabolomics using ultrahigh-pressure liquid chromatography coupled with tandem mass spectrometry. Subsequent analyses, including partial least squares-discriminant analysis modeling, t tests and weighted metabolite coexpression network analysis (WMCNA), were conducted to identify key differentially expressed metabolites (DEMs) and metabolite sets. Furthermore, we utilized least absolute shrinkage and selection operato and random fores analyses for biomarker prediction. The use of each metabolite and metabolite set to detect DILI was evaluated with area under the receiver operating characteristic curves. We found 247 differentially expressed salivary metabolites between the DILI group and the HC group. Using WMCNA, we identified a set of 8 DEMs closely related to liver injury for further prediction testing. Interestingly, the distinct separation of DILI patients and HCs was achieved with five metabolites, namely, 12-hydroxydodecanoic acid, 3-hydroxydecanoic acid, tetradecanedioic acid, hypoxanthine, and inosine (area under the curve: 0.733-1). Salivary metabolomics revealed previously unreported metabolic alterations and diagnostic biomarkers in the saliva of DILI patients. Our study may provide a potentially feasible and noninvasive diagnostic method for DILI, but further validation is needed.

Mechanism of intestinal microbiota disturbance promoting the occurrence and development of esophageal squamous cell carcinoma——based on microbiomics and metabolomics

Esophageal squamous cell carcinoma (ESCC) is a high-risk malignant tumor that has been reported in China. Some studies indicate that gut microbiota disorders can affect the occurrence and development of ESCC, but the underlying mechanism remains unclear. In this study, we aimed to explore the possible underlying mechanisms using microbiomics and metabolomics. Fifty ESCC patients and fifty healthy controls were selected as the study subjects according to sex and age, and fecal samples were collected. 16S rDNA sequencing and LC‒MS were used for microbiomics and nontargeted metabolomics analyses. We found significant differences in the composition of the gut microbiota and metabolites between the ESCC patients and control individuals (P < 0.05). ESCC patients exhibited increased abundances of Fusobacteriaceae and Lactobacillus, increased levels of GibberellinA34 and decreased levels of 12-hydroxydodecanoic acid; these metabolites could be diagnostic and predictive markers of ESCC. An increase in the abundance of Enterobacteriaceae and Lactobacillus significantly reduced the content of L-aspartate and pantothenic acid, which may be involved in the occurrence and development of ESCC by downregulating the expression of proteins in the pantothenate and coenzyme A biosynthesis pathways. An imbalance in the intestinal flora may decrease the number of eosinophils in peripheral blood, resulting in the activation of an inflammatory response and immune dysfunction, leading to ESCC deterioration. We hypothesize that this imbalance in the gut microbiota can cause an imbalance in intestinal metabolites, which can activate carcinogenic metabolic pathways, affect inflammation and immune function, and play a role in the occurrence and development of ESCC.

Plasma metabolomics and proteomics reveal novel molecular insights and biomarker panel for cholelithiasis

BackgroundCholelithiasis is a gastrointestinal disease that is associated with the highest socioeconomic cost. A diagnosis of cholelithiasis based on clinical features is significantly limited, and direct molecular insights into cholelithiasis and the relationship between cholelithiasis and clinical biochemical parameters are unclear. ObjectivesUncovering direct molecular insights into cholelithiasis and the relationship between cholelithiasis and clinical biochemical parameters. Identifying sensitive and specific biomarkers for this disease. MethodsParallel metabolomic and proteomic analyses of plasma from cholelithiasis patients (CPs) and healthy control individuals (HCs) without cholelithiasis were performed using ultrahigh-performance liquid chromatography-tandem mass spectrometry. A multimodule coexpression network analysis and integrated machine learning methods, including least absolute shrinkage and selection operator, random forest, and support vector machine, were used for bioinformatic analyses. An independent cohort and the cross-validation of the combination of two cohorts were used to evaluate the diagnostic performance of the panel. ResultsArachidonic acid metabolism was significantly different between the CP and HC groups. Glyceraldehyde-3-phosphate dehydrogenase, actin beta, phosphoglycerate mutase 1, Enolase 1, Myeloperoxidase, and actin alpha 1 were identified as potential proteins related to cholelithiasis. The correlation between the merged modules and clinical biochemical tests was calculated. A diagnostic panel composed of four candidate biomarkers, including 3-oxotetradecanoic acid, 12-hydroxydodecanoic acid, hemoglobin subunit delta (HBD), and fibrinogen beta chain (FGB), was proposed based on three modules that were significantly associated with cholelithiasis. The classification according to the diagnostic panel detected CPs with an area under the curve (AUC) of 0.955. External validation in an independent cohort resulted in similar accuracy (AUC=0.995). ConclusionsThis study provided some direct molecular insights into cholelithiasis by showing the differences in plasma metabolic and protein profiles between CPs and HCs and presented a potential biomarker panel with two metabolites (3-oxotetradecanoic acid, 12-hydroxydodecanoic acid) and two proteins (HBD, FGB) for predicting cholelithiasis. We also explored the potential correlation of clinical biochemical parameters with combined modules. These findings may provide some reference for the diagnosis of cholelithiasis in clinical practice.

Combined analysis of the metabolome and transcriptome reveals candidate genes involved in lipid metabolism in Heuchera micrantha Douglas ex Lindl.

Heuchera micrantha is widely used as a ornamental plant due to its strong environmental adaptability. The lipid metabolites in its leaves have obvious effects on biotic and abiotic stresses. However, the molecular mechanisms underlying lipid metabolism remain poorly understood. A total of 125 lipid metabolites were obtained from the four varieties, of which 109 were common to all four varieties, while very few (a maximum of four) were unique to a single variety. All these lipid metabolites could be classified into one of the following five classes: glycerol ester, phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, and free fatty acids. Most could be classified as free fatty acids. Principal component analysis separated the four varieties into three groups. A total of 79 differential lipid metabolites were identified, with γ-linolenic acid, α-linolenic acid, 12-hydroxydodecanoic acid, lysoPC 18:3 (2n isomer), lysoPE 16:0 (2n isomer), and lysoPE 16:0 being the main differential lipid metabolites among the four varieties. Transcriptome data from the four varieties revealed a total of 1733 differentially expressed genes (DEGs), which could be annotated into 15 KEGG pathways related to lipid metabolism. K-means clustering analysis of differential lipid metabolites and DEGs identified a total of 12 key candidate genes: 1 AOC, 1 new gene, 1 PYCT, 1 DPP, 1 KASII, 1 LCAT, 1 AKR, 1 CYP94A5, 1 GLB, 2 LACS, and 1 ACOX3. The AOC gene plays an important role in the synthesis of α-linolenic acid and γ-linolenic acid. KASII and LACS mainly control the levels of free fatty acids, while AKR and DPP affect the glycerol ester content. Taken together, these results provide insight into the molecular mechanism of lipid metabolism and provide valuable information about the formation of lipid metabolites in H. micrantha.

Comparison of lauric acid and 12-hydroxylauric acid in the alleviation of drought stress in peach (Prunus persica (L.) Batsch).

Water shortage is a key factor that can restrict peach tree growth. Plants produce fatty acids and the fatty acid derivatives lauric acid (LA) and 12-hydroxylauric acid (LA-OH), which are involved in abiotic stress responses, but the underlying stress response mechanisms remain unclear. In this study, physiological examination revealed that in Prunus persica (L.) Batsch, pretreatment with 50 ppm LA-OH and LA reduced drought stress, efficiently maintained the leaf relative water content, and controlled the relative conductivity increase. Under drought stress, LA-OH and LA treatments prevented the degradation of photosynthetic pigments, increased the degree of leaf stomatal opening and enhanced the net photosynthetic rate. Compared with drought stress, LA-OH and LA treatment effectively increased the net photosynthetic rate by 204.55% and 115.91%, respectively, while increasing the Fv/Fm by 2.75% and 7.75%, respectively, but NPQ decreased by 7.67% and 37.54%, respectively. In addition, the level of reactive oxygen species increased under drought stress. The content of O2 - in LA-OH and LA treatment decreased by 12.91% and 11.24% compared to CK-D, respectively, and the content of H2O2 decreased by 13.73% and 19.94%, respectively. At the same time, the content of malondialdehyde (MDA) decreased by 55.56% and 58.48%, respectively. We believe that the main reason is that LA-OH and LA treatment have improved the activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). The application of exogenous LA increased the levels of soluble sugars, soluble proteins, proline and free amino acids under drought stress, and maintained the osmotic balance of cells. Compared with CK-D treatment, it increased by 24.11%, 16.89%, 29.3% and 15.04%, respectively. At the same time, the application of exogenous LA-OH also obtained similar results. In conclusion, exogenous LA-OH and LA can alleviate the damage to peach seedlings caused by drought stress by enhancing the photosynthetic and antioxidant capacities, increasing the activities of protective enzymes and regulating the contents of osmotic regulators, but the molecular mechanism is still in need of further exploration.

Metabonomics reveals the main small molecules differences between green and white egg shells in ducks

The eggshell colour is related to the biological functions of birds, and the colour of poultry eggshells can affect consumers' choices. This study explored the difference in the metabolite composition of duck eggshells to screen the key substances that affect the eggshell colours. The green and white duck eggshells were selected for non-targeted metabolomics analysis. We screened 402 and 512 differentially expressed metabolites in the negative and positive ion modes, respectively. Among them, 40 differentially expressed metabolites were annotated with specific names and related functions, of which the expression levels of 8 metabolites showed extremely significant differences. They were 2-heptanone, 12-hydroxydodecanoic acid, D-fructose, dodecanedioic acid, L-leucine, methyl jasmonate, palmitoleic acid, and styrene oxide. Additionally, the annotated differentially expressed metabolites were enriched in 33 metabolic pathways, including aminoacyl-tRNA biosynthesis, amino acid metabolism, galactose metabolism, etc. The results showed that the expression of metabolites between green and white eggshells differed in ducks; among metabolites with extremely significant differences in expression, the expression level of L-leucine in green eggshells was higher than that in ducks white eggshells. Therefore, we speculated that the increased expression of L-leucine promoted the response of related metabolic pathways, enhanced the expression of antioxidants, and changed the eggshell colours. Highlights The metabolites of green and white shells were different. 40 DEMs were annotated with specific names and related functions. The annotated DEMs were enriched in 33 metabolic pathways.

Metabolomics study reveals the alteration of fatty acid oxidation in the hearts of diabetic mice by empagliflozin.

Empagliflozin (Empa, SGLT2 inhibitor) is widely used in clinical situations for the management of diabetes. It has beneficial effects in reducing cardiac dysfunction and heart failure. However, rare studies have reported the potential mechanisms of Empa reaction. Here, we treated db/db diabetic mice with Empa and collected the heart tissue for metabolomics study. We found that db/db mice showed obvious differences in metabolomics profile compared with db/m mice. Many amino acid metabolism pathways and glycerophospholipids and fatty acyl carnitines were significantly enriched in db/db mice. Detailed analysis revealed the alteration of fatty acid oxidation in db/db mice. Interestingly, many metabolites in the fatty acid oxidation pathway, such as myristoleic acid, 12-hydroxydodecanoic acid, (15-carboxypentadecanoyl)carnitine, decanoylcarnitine, and propionylcarnitine, were significantly rescued by Empa treatment. These results suggest that fatty acid oxidation is one of the targets for Empa treatment in the heart of db/db mice. This study provided new possibilities for the development of therapeutic interventions for diabetic cardiomyopathy.

Biochemical analysis of leptospiral LPS explained the difference between pathogenic and non-pathogenic serogroups

Lipopolysaccharide (LPS) is the major surface antigen of Leptospira. In this study, the genes involved in the LPS biosynthesis were analyzed and compared by bioinformatics tools. Also, the chemical composition analysis of leptospiral lipopolysaccharides (LPS) extracted from 5 pathogenic serovars like Autumnalis, Australis, Ballum, Grippotyphosa, Pomona, and the nonpathogenic serovar Andamana was performed. Methods used were Limulus amebocyte lysate assay (LAL), gas chromatography-mass spectrometry (GC-MS), fourier transform infrared spectroscopy (FT-IR), and nuclear magnetic resonance spectroscopy (NMR). LAL assay showed a significantly higher level of endotoxicity among pathogenic serovars (~0.490 EU/mL) than that of nonpathogenic Andamana (~0.102 EU/mL). FAMES analysis showed the presence of palmitic acid (C16:0), hydroxy lauric acid (3-OH-C12:0), and oleic acid (C18:0). Palmitoleic acid (C16: 1), and 3- hydroxy palmitate (3-OH-C16:0) was detected only in pathogenic serovars. In contrast myristoleic acid (C14:1) and stearic acid (C18:0) were present in Andamana. FTIR analysis revealed C–O–C stretch of esters, 3°ROH functional groups and carbohydrate vibration range were similar among pathogenic serovars. The NMR analysis reveals similarity for 6 deoxy sugars and methyl groups of Autumnalis, Australis, and Ballum. Further, the presence of palmitoleic acid and 3-hydroxy palmitate may be the significant pathogen-associated predisposing factor. This mediates high osmolarity glycerol (HOG) mediated stress response in leptospiral LPS mediated pathogenesis.

Ultra-low switching reverse mode liquid crystal gels.

This research investigates the electro-optical properties of reverse mode liquid crystal gel (LC-gel) scattering films. The LC-gel has been fabricated through the fibrous self-assembly of the gelator 12-hydroxydodecanoic acid (G12) and mesogen monomer (RM257) in nematic LC HTW106700-100 (HTW). Adding RM257 monomer improves the transparency in the OFF state and enhances scattering effects in the ON state. Moreover, an extremely low switching voltage (∼ 1 V) is demonstrated.

Whole-Cell Biotransformation of 1,12-Dodecanedioic Acid from Coconut Milk Factory Wastewater by Recombinant CYP52A17SS Expressing Saccharomyces cerevisiae

Biotransformation of fatty acids from renewable wastewater as feedstock to value-added chemicals is a fascinating commercial opportunity. α,ω-Dicarboxylic acids (DCAs) are building blocks in many industries, such as polymers, cosmetic intermediates, and pharmaceuticals, and can be obtained by chemical synthesis under extreme conditions. However, biological synthesis can replace the traditional chemical synthesis using cytochrome P450 enzymes to oxidize fatty acids to DCAs. Saccharomyces cerevisiae BY(2R)/pYeDP60-CYP52A17SS (BCM), a transgenic strain expressing the galactose-inducible CYP52A17SS cytochrome P450 enzyme, was able to grow in a coconut milk factory wastewater (CCW) medium and produced 12-hydroxydodecanoic acid (HDDA) and 1,12-dodecanedioic acid (DDA). The supplementation of CCW with 10 g/L yeast extract and 20 g/L peptone (YPCCW) markedly increased the yeast growth rate and the yields of 12-HDDA and 1,12-DDA, with the highest levels of approximately 60 and 38 µg/L, respectively, obtained at 30 °C and pH 5. The incubation temperature and medium pH strongly influenced the yeast growth and 1,12-DDA yield, with the highest 1,12-DDA formation at 30 °C and pH 5–5.5. Hence, the S. cerevisiae BCM strain can potentially be used for producing value-added products from CCW.

Characterization of antifungal metabolites produced by Lactobacillus plantarum and Lactobacillus coryniformis isolated from rice rinsed water.

A recent spike in demand for chemical preservative free food has derived the scientific community to develop natural ways of food preservation. Therefore, bio-preservation could be considered as the great alternative over chemical ones owing to its potential to increase shelf-life and nutritional values of foodstuffs. In the present study, lactic acid producing bacterial species were isolated from rice rinsed water and identified by 16S rRNA gene sequencing as Lactobacillus plantarum BCH-1 (KX388380) and Lactobacillus coryniformis BCH-4 (KX388387). Antifungal metabolites from both Lactobacillus species were extracted by polarity-based solvents in which ethyl acetate showed remarkable antifungal activity against Aspergillus flavus and Aspergillus fumigatus by disc diffusion assay. Different organic acids and fatty acids have been identified by reversed-phase high-performance liquid chromatography (RP-HPLC) and gas chromatography-mass spectrometry (GC-MS) analysis, respectively. Lactic acid and citric acid were the major organic acids found in ethyl acetate fractions of L. plantarum and L. coryniformis, respectively. Similarly, 9,12-otadecadienoic acid (Z,Z)-methyl ester and hexadecanoic acid, methyl ester were the major fatty acids found in n-hexane fractions of L. plantarum and L. coryniformis respectively. Moreover, the isolation of novel antifungal metabolites from locally isolated Lactobacillus species was focused and it was revealed that organic acids are important contributors towards antifungal potential. A novel fatty acid (i.e. 12-hydroxydodecanoic acid) has also been explored and found as potential metabolite against filamentous fungi. Conclusively, various metabolites isolated from non-dairy source showed antifungal activity especially against Aspergillus species. Hence, these metabolites have been considered as a good choice for bio-preservation.

Biotransformation of lauric acid into 1,12-dodecanedioic acid using CYP52A17 expressed in Saccharomyces cerevisiae and its application in refining coconut factory wastewater

1,12-Dodecanedioic acid (DDA), a primary compound and an intermediate precursor for various chemical products, is normally produced by chemical synthesis, which presents potential disadvantages. Instead, the biosynthesis of 1,12-DDA by recombinant (r) microorganisms may offer a viable production route. Cytochrome P450 (CYP) can terminally oxidize fatty acids to ω-hydroxy-fatty acids and further to dicarboxylic acids (ω-oxidation). The wild type (r) CYP52A17LL and its engineered L261S/L490S form, in which the two leucine residues were changed into serine (rCYP52A17SS), were expressed in Pichia pastoris and a strain of Saccharomyces cerevisiae coexpressing the yeast NADPH cytochrome P450 reductase gene. Both organisms produced 12-hydroxydodecanoic acid (HDDA) and 1,12-DDA from lauric acid. In vitro, microsomes containing rCYP52A17SS extracted from the S. cerevisiae strain BY(2R)/pYeDP60-CYP52A17SS were able to convert lauric acid to 12-HDDA quite efficiently. Biotransformation of lauric acid using S. cerevisiae BY(2R)/pYeDP60-CYP52A17SS in culture gave the highest level of 12-HDDA (45.8 μM) at 24 h, which was oxidized to yield 20.8 μM of 1,12-DDA at 72 h. The recombinant S. cerevisiae BY(2R)/pYeDP60-CYP52A17SS, which was initially cultured in YPGE, produced the highest yield of 1,12-DDA from coconut milk wastewater at 24 h. Hence, our designed S. cerevisiae strain BY(2R)/pYeDP60-CYP52A17SS can potentially produce 1,12-DDA for industrial applications.

Enzymatic Synthesis of ω-Hydroxydodecanoic Acid By Employing a Cytochrome P450 from Limnobacter sp. 105 MED

ω-Hydroxylated fatty acids are valuable and versatile building blocks for the production of various adhesives, lubricants, cosmetic intermediates, etc. The biosynthesis of ω-hydroxydodecanoic acid from vegetable oils is one of the important green pathways for their chemical-based synthesis. In the present study, the novel monooxygenase CYP153AL.m from Limnobacter sp. 105 MED was used for the whole-cell biotransformations. We constructed three-component system that was comprised of CYP153AL.m, putidaredoxin and putidaredoxin reductase from Pseudomonas putida. This in vivo study demonstrated that CYP153AL.m is a powerful catalyst for the biosynthesis of ω-hydroxydodecanoic acid. Under optimized conditions, the application of a solid-state powdered substrate rather than a substrate dissolved in DMSO significantly enhanced the overall reaction titer of the process. By employing this efficient system, 2 g/L of 12-hydroxydodecanoic acid (12-OHDDA) was produced from 4 g/L of its corresponding fatty acid, which was namely dodecanoic acid. Furthermore, the system was extended to produce 3.28 g/L of 12-OHDDA using 4 g/L of substrate by introducing native redox partners. These results demonstrate the utility of CYP153AL.m-catalyzed biotransformations in the industrial production of 12-OHDDA and other valuable building blocks.

A metabolomics approach to characterize raw, pasteurized, and ultra-high temperature milk using ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry and multivariate data analysis

We developed a metabolomics workflow using ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry to determine the effect of thermal treatment on milk composition and metabolites based on multivariate data analysis. We analyzed raw, pasteurized, and UHT milk samples. The samples were first centrifuged to remove the fat layer and mixed with methanol to precipitate proteins. Subsequently, the supernatant was analyzed by ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry in electrospray negative mode. Mass spectral data were acquired in MSE mode, a technique whereby both precursor and fragment mass spectral are simultaneously acquired by alternating between low and high collision energy (CE) during a single analytical run, to enable metabolite identification. Based on multivariate data analysis, these markers were significantly affected by thermal treatment. Among the 8 potential markers, we identified 7 oxylipids (9-hydroxydecanoic acid, 12-hydroxydodecanoic acid, 2-hydroxymyristic acid, 3-hydroxytetradecanoic acid, 5-hydroxyeicosatetraenoic acid, 3-hydroxyhexadecanoic acid, and 10-hydroxyoctadecanoic acid) and 1 phospholipid (LysoPE, hexadecanoyl-lysophosphatidylethanolamine). The oxylipids seemed to be adequate for distinguishing UHT milk from raw and pasteurized milk. The structures of the 8 potential markers were identified and characterized using informatics software. Our metabolomics workflow provides a fast approach for the identification of various types of milk.

Cytochrome P450 4A11 inhibition assays based on characterization of lauric acid metabolites

This study was designed to characterize lauric acid metabolism to facilitate the establishment of cytochrome P450 4A11 (CYP4A11) inhibition assay. Three metabolites (2-, 11-, and 12-hydroxylauric acids) were identified in pooled human liver microsomes based on comparisons with authentic standards. Reaction phenotyping using 14 recombinant CYPs showed that ω-hydroxylation was mediated dominantly by CYP4A11 and marginally by CYP4F3B. CYP2B6 played an exclusive role in the formation of 2-hydroxylauric acid. The production of 11-hydroxylauric acid was mediated by CYP2E1, CYP2C9, CYP2B6, CYP1A2, CYP3A4, and CYP4A11. The IC50 values of HET0016, a well-known pan-CYP4 inhibitor, against the formation of 12-, 11-, and 2-hydroxylauric acid were 1.0, 1.0, and 0.009 μM, respectively. Among the 50 natural compounds examined, plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) inhibited the formation of 12-, 11-, and 2-hydroxylauric acid with IC50 values of 1.7, 2.3, and 2.7 μM, respectively. In the selectivity study, HET0016 inhibited CYP2B6 with an IC50 of 9.2 nM, as well as CYP1A2, CYP2C19, and CYP2E1 with IC50 values of 1–2 μM. Plumbagin inhibited all CYP enzymes tested with IC50 values of 1.7–3.0 μM. These methods can be used as tools to develop CYP4A11 inhibitors; simultaneous determination of the hydroxylauric acid metabolites provides further information on selectivity.

In vitro oxidative decarboxylation of free fatty acids to terminal alkenes by two new P450 peroxygenases

BackgroundP450 fatty acid decarboxylases represented by the unusual CYP152 peroxygenase family member OleTJE have been receiving great attention recently since these P450 enzymes are able to catalyze the simple and direct production of 1-alkenes for potential applications in biofuels and biomaterials. To gain more mechanistic insights, broader substrate spectra, and improved decarboxylative activities, it is demanded to discover and investigate more P450 fatty acid decarboxylases.ResultsHere, we describe for the first time the expression, purification, and in vitro biochemical characterization of two new CYP152 peroxygenases, CYP-Aa162 and CYP-Sm46Δ29, that are capable of decarboxylating straight-chain saturated fatty acids. Both enzymes were found to catalyze the decarboxylation and hydroxylation of a broad range of free fatty acids (C10–C20) with overlapping substrate specificity, yet distinct chemoselectivity. CYP-Sm46Δ29 works primarily as a fatty (lauric) acid decarboxylase (66.1 ± 3.9% 1-undecene production) while CYP-Aa162 more as a fatty (lauric) acid hydroxylase (72.2 ± 0.9% hydroxy lauric acid production). Notably, the optical spectroscopic analysis of functional CYP-Sm46Δ29 revealed no characteristic P450 band, suggesting a unique heme coordination environment. Active-site mutagenesis analysis showed that substitution with the proposed key decarboxylation-modulating residues, His85 and Ile170, enhanced the decarboxylation activity of CYP-Aa162 and P450BSβ, emphasizing the importance of these residues in directing the decarboxylation pathway. Furthermore, the steady-state kinetic analysis of CYP-Aa162 and CYP-Sm46Δ29 revealed both cooperative and substrate inhibition behaviors which are substrate carbon chain length dependent.ConclusionsOur data identify CYP-Sm46Δ29 as an efficient OleTJE-like fatty acid decarboxylase. Oxidative decarboxylation chemoselectivity of the CYP152 decarboxylases is largely dependent upon the carbon chain length of fatty acid substrates and their precise positioning in the enzyme active site. Finally, the kinetic mode analysis of the enzymes could provide important guidance for future process design.

Expression of CYP4V2 in human THP1 macrophages and its transcriptional regulation by peroxisome proliferator-activated receptor gamma

Because macrophages respond to a variety of pathological and pharmacological reagents, understanding the role of P450s in macrophages is important for therapeutic intervention. There has been a lack of research on CYP4 in macrophages, but fatty acid accumulation and lipid trafficking in macrophages have been suggested to be a main cause of atherosclerosis. All human CYP4 genes (n=12) were screened in THP1 macrophages by gene-specific reverse transcriptase-polymerase chain reaction (RT-PCR). Only CYP4V2 exhibited strong expression of both mRNA and protein. Expression levels of both CYP4V2 mRNA and protein were significantly reduced after treatment with peroxisome proliferator-activated receptor gamma (PPARγ) antagonist GW9662. However, the expression levels of CYP4V2 were not changed by PPARα antagonist (GW6471) and liver X receptor alpha antagonist (22-S hydroxycholesterol). A metabolite of the CYP4V2 enzyme, 12-hydroxydodecanoic acid, was detected in THP1 macrophages, and this metabolite was significantly decreased after treatment with the PPARγ inhibitor GW9662 (>80% decreased, p<0.05). In summary, fatty acid metabolizing protein CYP4V2 was identified in human THP1 macrophages, and its expression was regulated by PPARγ. Further study is required to understand the role of CYP4V2 with regard to fat accumulation in the activated macrophage and atherosclerotic plaque development.

New Poly(Hydroxylauric-co-Lactic Acid) Liquid Polymer for Dissolving Lipophilic Drugs

A liquid, biocompatible polyester based polymer, which could facilitate injectable formulations by simple mixing with the active substance (drug) is much needed by the pharmaceutical companies. A favourite candidate is polylactic acid (PLA) which is biocompatible and biodegradable. However PLA is solid with high crystallinity. Thus, in this research, hydroxylauric acid (HOLA) was copolymerized with lactic acid (LA) in different ratios by polycondensation technique at 180 °C, without a metal catalyst and avoiding the formation of interfering lactides, to provide a liquid polyester. The copolymers molecular weights were determined by Gel Permeation Chromatography (GPC) and their physical states indicated as solid or liquid were noted. The structures as polyesters were confirmed by FT-IR and 1H NMR spectroscopy. Poly(HOLA:LA) products from reactant ratios 0:100 is solid, while ratios of 20:80, 40:60 are mixed (paste) and 60:40, 80:20 and 100:0 are liquids. Thus, the liquid polyesters from the polycondensation of HOLA and LA without catalyst were picked as potential candidates for dissolving hydrophobic drugs that could be used as injectables in controlled drug delivery experiments.

Structural profiling of wax biopolymer from Pinus roxburghii Sarg. needles using spectroscopic methods

Pinus roxburghii Sarg. is the most abundant species in Himalayan region. The needles of the species largely contribute to the forest biomass and remain the major cause of forest fires leading to climate change, biodiversity loss, etc. Intriguingly, the layer of needles contains wax, a biomacromolecule with potential chemical functionalities for value addition. In the present study, a distinctive approach towards complete structural analysis of the isolated wax in its native state has been done using 1H, 13C, HSQC, HMBC, COSY, TOCSY along with GC–MS of the methyl esters of constituent fatty acids. The wax was isolated in a quantitative yield of 1.64% and analyses suggest that it is a polymer of linearly attached fatty acid esters which on hydrolysis yielded three types of ω-hydroxy fatty acids viz. 12-hydroxydodecanoic acid, 14-hydroxytetradecanoic acid and 16-hydroxyhexadecanoic acid in a ratio of 1:1:2 respectively. Complete assignments for a carbonyl group, α-, β- and other methylenes present in wax were achieved; corroborating the presence of polyester. In particular, identification of wax structure was accomplished through NMR; thereby providing a lead towards future structural analysis of waxes in their native form. The study would also be helpful to generate commercially important compounds derived from pine needle wax. This will offer an opportunity for utilisation of pine needle biomass: a root cause of Himalayan forest fires.