Pregnenolone and progesterone production from natural sterols using recombinant strain of Mycolicibacterium smegmatis mc2 155 expressing mammalian steroidogenesis system.

BACKGROUND: Pregnenolone and progesterone are the life-important steroid hormones regulating essential vital functions in mammals, and widely used in different fields of medicine. Microbiological production of these compounds from sterols is based on the use of recombinant strains expressing the enzyme system cholesterol hydroxylase/C20-C22 lyase (CH/L) of mammalian steroidogenesis. However, the efficiency of the known recombinant strains is still low. New recombinant strains and combination approaches are now needed to produce these steroid hormones. RESULTS: Based on Mycolicibacterium smegmatis, a recombinant strain was created that expresses the steroidogenesis system (CYP11A1, adrenodoxin reductase, adrenodoxin) of the bovine adrenal cortex. The recombinant strain transformed cholesterol and phytosterol to form progesterone among the metabolites. When 3-methoxymethyl ethers of sterols were applied as bioconversion substrates, the corresponding 3-ethers of pregnenolone and dehydroepiandrosterone (DHEA) were identified as major metabolites. Under optimized conditions, the recombinant strain produced 85.2 ± 4.7 mol % 3-methoxymethyl-pregnenolone within 48 h, while production of 3-substituted DHEA was not detected. After the 3-methoxymethyl function was deprotected by acid hydrolysis, crystalline pregnenolone was isolated in high purity (over 98%, w/w). The structures of steroids were confirmed using TLC, HPLC, MS and 1H- and 13C-NMR analyses. CONCLUSION: The use of mycolicybacteria as a microbial platform for the expression of systems at the initial stage of mammalian steroidogenesis ensures the production of valuable steroid hormones-progesterone and pregnenolone from cholesterol. Selective production of pregnenolone from cholesterol is ensured by the use of 3-substituted cholesterol as a substrate and optimization of the conditions for its bioconversion. The results open the prospects for the generation of the new microbial biocatalysts capable of effectively producing value-added steroid hormones.

Acylation of the Rat Brain Proteins is Affected by the Inhibition of Pyruvate Dehydrogenase in vivo.

Organism adaptation to metabolic challenges requires coupling of metabolism to gene expression. In this regard, acylations of histones and metabolic proteins acquire significant interest. We hypothesize that adaptive response to inhibition of a key metabolic process, catalyzed by the acetyl-CoA-generating pyruvate dehydrogenase (PDH) complex, is mediated by changes in the protein acylations. The hypothesis is tested by intranasal administration to animals of PDH-specific inhibitors acetyl(methyl)phosphinate (AcMeP) or acetylphosphonate methyl ester (AcPMe), followed by the assessment of physiological parameters, brain protein acylation, and expression/phosphorylation of PDHA subunit. At the same dose, AcMeP, but not AcPMe, decreases acetylation and increases succinylation of the brain proteins with apparent molecular masses of 15-20 kDa. Regarding the proteins of 30-50 kDa, a strong inhibitor AcMeP affects acetylation only, while a less efficient AcPMe mostly increases succinylation. The unchanged succinylation of the 30-50 kDa proteins after the administration of AcMeP coincides with the upregulation of desuccinylase SIRT5. No significant differences between the levels of brain PDHA expression, PDHA phosphorylation, parameters of behavior or ECG are observed in the studied animal groups. The data indicate that the short-term inhibition of brain PDH affects acetylation and/or succinylation of the brain proteins, that depends on the inhibitor potency, protein molecular mass, and acylation type. The homeostatic nature of these changes is implied by the stability of physiological parameters after the PDH inhibition.

Expression of Synthetic cyp102A1-LG23 Gene and Functional Analysis of Recombinant Cytochrome P450 BM3-LG23 in the Actinobacterium Mycolicibacterium smegmatis.

Cytochrome CYP102A1 (P450 BM3) of Priestia megaterium (bas. Bacillus megaterium) has several unique functional features and thus provides an ideal object for directed evolution and other synthetic applications. Previously, the CYP102A1-LG23 mutant with 14 mutations in the heme part was obtained that hydroxylates several androstanes at C7ß with the formation of products with the anti-inflammatory and neuroprotective activities. In this study, synthetic cyp102A1-LG23 gene encoding the P450 BM3 mutant was expressed as a component of either monocistronic operon or bicistronic operon containing the gdh (glucose dehydrogenase, GDH) or zwf2 (glucose 6-phosphate dehydrogenase, G6PD) gene in Mycolicibacterium smegmatis BD cells. The recombinant bacteria were able hydroxylate androst-4-ene-3,17-dione (AD) into 7ß-OH-AD. Their biocatalytic activity was increased twice by increasing the solubility of CYP102A1-LG23 protein in the cells and supplementing the cells with the additional cofactor regeneration system by introducing GDH and G6PD. The maximum 7ß-OH-AD yield (37.68 mol%) was achieved by co-expression of cyp102A1-LG23 and gdh genes in M. smegmatis. These results demonstrate the possibility of using synthetic genes to obtain recombinant enzymes and expand our understanding of the processes involved in steroid hydroxylation by bacterial cytochromes. The data obtained can be used to develop new approaches for microbiological production of 7ß-hydroxylated steroids in genetically modified Mycolicibacterium species.

Identification of a Gene Encoding a New Fungal Steroid 7-Hydroxylase and Its Functional Characterization in Pichia pastoris Yeast.

The hydroxylation of steroids in the C7ß position is one of the rare reactions that allow the production of value-added precursors in the synthesis of ursodeoxycholic acid and other pharmaceuticals. Recently, we discovered this activity in the ascomycete Curvularia sp. VKM F-3040. In this study, the novel gene of 7-hydroxylase (P450cur) was identified as being heterologously expressed and functionally characterized in Pichia pastoris. Transcriptome data mining and differential expression analysis revealed that 12 putative genes in Curvularia sp. mycelia significantly increased their expression in response to dehydroepiandrosterone (DHEA). The transcriptional level of the most up-regulated cytochrome P450cur gene was increased more than 300-fold. A two-gene construct with a candidate P450cur gene and the gene of its natural redox partner, NADPH-cytochrome P450 reductase (CPR), which is interconnected by a T2A element, was created. Using this construct, recombinant P. pastoris strains co-expressing fungal P450cur and CPR genes were obtained. The functional activity of the recombinant P450cur was studied in vivo during the bioconversion of androstane steroids. The fungal 7-monooxygenase predominantly catalyzed the 7ß-hydroxylation of androstadienedione (ADD), DHEA, and androstenediol, whereas 1-dehydrotestosterone was hydroxylated by P450cur mainly at the C7-Hα position. To our knowledge, this is the first report of a recombinant yeast capable of catalyzing the 7α/ß-hydroxylation of ADD and DHEA.

Hydroxylation of pregnenolone and dehydroepiandrosterone by zygomycete Backusella lamprospora VKM F-944: selective production of 7α-OH-DHEA.

In this paper, we studied the transformation of two 3ß-hydroxy-5-ene-steroids-pregnenolone and dehydroepiandrosterone (DHEA) by Backusella lamprospora VKM F- 944. The soil-dwelling zygomycete wild-type strain has been earlier selected during the screening and previously unexplored for this purpose. The fungus fully converted pregnenolone to form a mixture of axial 7α-hydroxy-pregnenolone and 7α,11α-dihydroxy-pregnenolone, while no metabolites with ß-orientation of the hydroxyl group were detected. The pathway to 7α,11α-diOH-pregnenolone seems to include 7α-hydroxylation of 11α-hydroxylated derivative. The only product from DHEA was identified as 7α-hydroxy-DHEA. The structures of steroid metabolites were confirmed by HPLC, mass-spectrometry (MS), and 1H and 13C NMR analyses. Under the optimized conditions, the yield of 7α-OH-DHEA reached 94% (w/w) or over 14 g/L in absolute terms, even at high concentration of the substrate (DHEA) (15 g/L). To our knowledge, it is the highest yield of the value-added 7α-OH-DHEA reported so far. The results contribute to the knowledge of the diversity of the wild-type fungal strains capable of effective steroid hydroxylation. They could be applied for the production of allylic steroid 7α-alcohols that are widely used in medicine. KEY POINTS: • Zygomycete Backusella lamprospora actively hydroxylates 3ß-hydroxy-5-en-steroids. • Axial 7α-hydroxylation is the preferable reaction by the strain towards pregnenolone and DHEA. • The strain selectively produces 7α-OH-DHEA even at high substrate concentrations (up to 15 g/L).

Recombinant Extracellular Cholesterol Oxidase from Nocardioides simplex.

Cholesterol oxidase is a highly demanded enzyme used in medicine, pharmacy, agriculture, chemistry, and biotechnology. It catalyzes oxidation of 3ß-hydroxy-5-ene- to 3-keto-4-ene- steroids with the formation of hydrogen peroxide. Here, we expressed 6xHis-tagged mature form of the extracellular cholesterol oxidase (ChO) from the actinobacterium Nocardioides simplex VKM Ac-2033D (55.6 kDa) in Escherichia coli cells. The recombinant enzyme (ChONs) was purified using affinity chromatography. ChONs proved to be functional towards cholesterol, cholestanol, phytosterol, pregnenolone, and dehydroepiandrosterone. Its activity depended on the structure and length of the aliphatic side chain at C17 atom of the steroid nucleus and was lower with pregnenolone and dehydroepiandrosterone. The enzyme was active in a pH range of 5.25÷6.5 with the pH optimum at 6.0. Kinetic assays and storage stability tests demonstrated that the characteristics of ChONs were generally comparable with or superior to those of commercial ChO from Streptomyces hygroscopicus (ChOSh). The results contribute to the knowledge on microbial ChOs and evidence that ChO from N. simplex VKM Ac-2033D is a promising agent for further applications.

Phosphonate Inhibitors of Pyruvate Dehydrogenase Perturb Homeostasis of Amino Acids and Protein Succinylation in the Brain.

Mitochondrial pyruvate dehydrogenase complex (PDHC) is essential for brain glucose and neurotransmitter metabolism, which is dysregulated in many pathologies. Using specific inhibitors of PDHC in vivo, we determine biochemical and physiological responses to PDHC dysfunction. Dose dependence of the responses to membrane-permeable dimethyl acetylphosphonate (AcPMe2) is non-monotonous. Primary decreases in glutathione and its redox potential, methionine, and ethanolamine are alleviated with increasing PDHC inhibition, the alleviation accompanied by physiological changes. A comparison of 39 brain biochemical parameters after administration of four phosphinate and phosphonate analogs of pyruvate at a fixed dose of 0.1 mmol/kg reveals no primary, but secondary changes, such as activation of 2-oxoglutarate dehydrogenase complex (OGDHC) and decreased levels of glutamate, isoleucine and leucine. The accompanying decreases in freezing time are most pronounced after administration of methyl acetylphosphinate and dimethyl acetylphosphonate. The PDHC inhibitors do not significantly change the levels of PDHA1 expression and phosphorylation, sirtuin 3 and total protein acetylation, but increase total protein succinylation and glutarylation, affecting sirtuin 5 expression. Thus, decreased production of the tricarboxylic acid cycle substrate acetyl-CoA by inhibited PDHC is compensated by increased degradation of amino acids through the activated OGDHC, increasing total protein succinylation/glutarylation. Simultaneously, parasympathetic activity and anxiety indicators decrease.

Pd- and Cu-catalyzed approaches in the syntheses of new cholane aminoanthraquinone pincer-like ligands.

Cu- and Pd-catalyzed arylation of aminocholanes has been described for the first time. While this Cu-catalyzed protocol provides high yields in reactions of aminocholanes with iodoarenes, Pd catalysis was found to be preferable for the reactions of aminocholanes with dichloroanthraquinones. UV-vis titration of bis(cholanylamino)anthraquinones with a series of cations demonstrated their high binding affinity to Cu2+, Al3+, and Cr3+.

pH-Sensitive Liposomes with Embedded 3-(isobutylamino)cholan-24-oic Acid: What Is the Possible Mechanism of Fast Cargo Release?

pH-sensitive liposomes have great potential for biomedical applications, in particular as nanocontainers for the delivery of biologically active compounds to specific areas of the human body. In this article, we discuss the possible mechanism of fast cargo release from a new type of pH-sensitive liposomes with embedded ampholytic molecular switch (AMS, 3-(isobutylamino)cholan-24-oic acid) with carboxylic anionic groups and isobutylamino cationic ones attached to the opposite ends of the steroid core. AMS-containing liposomes demonstrated the rapid release of the encapsulated substance when altering the pH of an outer solution, but the exact mechanism of the switch action has not yet been accurately determined. Here, we report on the details of fast cargo release based on the data obtained using ATR-FTIR spectroscopy as well as atomistic molecular modeling. The findings of this study are relevant to the potential application of AMS-containing pH-sensitive liposomes for drug delivery.

Steroid modification by filamentous fungus Drechslera sp.: Focus on 7-hydroxylase and 17ß-hydroxysteroid dehydrogenase activities.

Fungal strain Drechslera sp. Ph F-34 was shown to modify 3-oxo- and 3-hydroxy steroids of androstane series to form the corresponding allylic 7-alcohols and 17ß-reduced derivatives thus evidencing the presence of 7α-, 7ß-hydroxylase and 17ß-hydroxysteroid dehydrogenase (17ß-HSD) activities. The growing mycelium predominantly hydroxylated androsta-1,4-diene-3,17-dione (ADD) at the 7ß-position, while much lower 7α-hydroxylation was observed. Along with 7ß-hydroxy-ADD and its corresponding 7α-isomer, their respective 17ß-alcohols were produced. In this study, transformation of ADD, androst-4-en-17ß-ol-3-one (testosterone, TS) and 3ß-hydroxyandrost-5-en-17-one (dehydroepiandrosterone, DHEA) by resting mycelium of Drechslera sp. have been estimated in different conditions with regard to the inducibility and functionality of the 17ß-HSD and 7-hydroxylase enzyme systems. Steroids of androstane, pregnane and cholane series were evaluated as inducers. The inhibitory analysis was provided using cycloheximide (CHX). Steroids were assayed using TLC and HPLC methods, and the structures were confirmed by mass-spectrometry, 1H and 13C NMR spectroscopy data. 17ß-HSD of the mycelium constitutively reduced 17-carbonyl group of ADD and DHEA to form the corresponding 17ß-alcohols, namely, androsta-1,4-diene-17ß-ol-3-one (1-dehydro-TS), and androst-5-ene-3ß,17ß-diol. Production of the 7α- and 7ß-hydroxylated derivatives depended on the induction conditions. The inducer effect relied on the steroid structure and decreased in the order: DHEA > pregnenolone > lithocholic acid. ß-Sitosterol did not induce hydroxylase activity in Drechslera sp. CHX fully inhibited the synthesis of 7-hydroxylase in Drechslera mycelium thus providing selective 17-keto reduction. Results contribute to the diversity of steroid modifying enzymes in fungi and can be used at the development of novel biocatalysts for production of valuable steroid 7(α/ß)- and 17ß-alcohols.

Increasing Inhibition of the Rat Brain 2-Oxoglutarate Dehydrogenase Decreases Glutathione Redox State, Elevating Anxiety and Perturbing Stress Adaptation.

Specific inhibitors of mitochondrial 2-oxoglutarate dehydrogenase (OGDH) are administered to animals to model the downregulation of the enzyme as observed in neurodegenerative diseases. Comparison of the effects of succinyl phosphonate (SP, 0.02 mmol/kg) and its uncharged precursor, triethyl succinyl phosphonate (TESP, 0.02 and 0.1 mmol/kg) reveals a biphasic response of the rat brain metabolism and physiology to increasing perturbation of OGDH function. At the low (TE)SP dose, glutamate, NAD+, and the activities of dehydrogenases of 2-oxoglutarate and malate increase, followed by their decreases at the high TESP dose. The complementary changes, i.e., an initial decrease followed by growth, are demonstrated by activities of pyruvate dehydrogenase and glutamine synthetase, and levels of oxidized glutathione and citrulline. While most of these indicators return to control levels at the high TESP dose, OGDH activity decreases and oxidized glutathione increases, compared to their control values. The first phase of metabolic perturbations does not cause significant physiological changes, but in the second phase, the ECG parameters and behavior reveal decreased adaptability and increased anxiety. Thus, lower levels of OGDH inhibition are compensated by the rearranged metabolic network, while the increased levels induce a metabolic switch to a lower redox state of the brain, associated with elevated stress of the animals.

Administration of Phosphonate Inhibitors of Dehydrogenases of 2-Oxoglutarate and 2-Oxoadipate to Rats Elicits Target-Specific Metabolic and Physiological Responses.

In vitro and in cell cultures, succinyl phosphonate (SP) and adipoyl phosphonate (AP) selectively target dehydrogenases of 2-oxoglutarate (OGDH, encoded by OGDH/OGDHL) and 2-oxoadipate (OADH, encoded by DHTKD1), respectively. To assess the selectivity in animals, the effects of SP, AP, and their membrane-penetrating triethyl esters (TESP and TEAP) on the rat brain metabolism and animal physiology are compared. Opposite effects of the OGDH and OADH inhibitors on activities of OGDH, malate dehydrogenase, glutamine synthetase, and levels of glutamate, lysine, citrulline, and carnosine are shown to result in distinct physiological responses. ECG is changed by AP/TEAP, whereas anxiety is increased by SP/TESP. The potential role of the ester moiety in the uncharged precursors of the 2-oxo acid dehydrogenase inhibitors is estimated. TMAP is shown to be less efficient than TEAP, in agreement with lower lipophilicity of TMAP vs. TEAP. Non-monotonous metabolic and physiological impacts of increasing OADH inhibition are revealed. Compared to the non-treated animals, strong inhibition of OADH decreases levels of tryptophan and beta-aminoisobutyrate and activities of malate dehydrogenase and pyruvate dehydrogenase, increasing the R-R interval of ECG. Thus, both metabolic and physiological actions of the OADH-directed inhibitors AP/TEAP are different from those of the OGDH-directed inhibitors SP/TESP, with the ethyl ester being more efficient than methyl ester.

Steroid Metabolism in Thermophilic Actinobacterium Saccharopolyspora hirsuta VKM Ac-666T.

The application of thermophilic microorganisms opens new prospects in steroid biotechnology, but little is known to date on steroid catabolism by thermophilic strains. The thermophilic strain Saccharopolyspora hirsuta VKM Ac-666T has been shown to convert various steroids and to fully degrade cholesterol. Cholest-4-en-3-one, cholesta-1,4-dien-3-one, 26-hydroxycholest-4-en-3-one, 3-oxo-cholest-4-en-26-oic acid, 3-oxo-cholesta-1,4-dien-26-oic acid, 26-hydroxycholesterol, 3ß-hydroxy-cholest-5-en-26-oic acid were identified as intermediates in cholesterol oxidation. The structures were confirmed by 1H and 13C-NMR analyses. Aliphatic side chain hydroxylation at C26 and the A-ring modification at C3, which are putatively catalyzed by cytochrome P450 monooxygenase CYP125 and cholesterol oxidase, respectively, occur simultaneously in the strain and are followed by cascade reactions of aliphatic sidechain degradation and steroid core destruction via the known 9(10)-seco-pathway. The genes putatively related to the sterol and bile acid degradation pathways form three major clusters in the S. hirsuta genome. The sets of the genes include the orthologs of those involved in steroid catabolism in Mycobacterium tuberculosis H37Rv and Rhodococcus jostii RHA1 and related actinobacteria. Bioinformatics analysis of 52 publicly available genomes of thermophilic bacteria revealed only seven candidate strains that possess the key genes related to the 9(10)-seco pathway of steroid degradation, thus demonstrating that the ability to degrade steroids is not widespread among thermophilic bacteria.

Biotransformation of androstenedione and androstadienedione by selected Ascomycota and Zygomycota fungal strains.

Filamentous fungi is a huge phylum of lower eukaryotes with diverse activities towards various substrates, however, their biocatalytic potential towards steroids remains greatly underestimated. In this study, more than forty Ascomycota and Zygomycota fungal strains of 23 different genera were screened for the ability to catalyze structural modifications of 3-oxo-androstane steroids, - androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD). Previously unexplored for these purposes strains of Absidia, Acremonium, Beauveria, Cunninghamella, Doratomyces, Drechslera, Fusarium, Gibberella genera were revealed capable of producing in a good yield valuable 7α-, 7ß-, 11α- and 14α-hydroxylated derivatives, as well as 17ß-reduced and 1(2)-dehydrogenated androstanes. The bioconversion routes of AD and ADD were proposed based on the key intermediates identification and time courses of the bioprocesses. Six ascomycete strains were discovered to provide effective 7ß-hydroxylation of ADD which has not been so far reported. The structures of major products and intermediates were confirmed by HPLC, mass-spectrometry (MS), 1H and 13C NMR analyses. The results contribute to the knowledge on the functional diversity of steroid-transforming filamentous fungi. Previously unexplored fungal biocatalysts capable of effective performing structural modification of AD and ADD can be applied for industrial bioprocesses of new generation.

Selective Inhibition of 2-Oxoglutarate and 2-Oxoadipate Dehydrogenases by the Phosphonate Analogs of Their 2-Oxo Acid Substrates.

Phosphonate analogs of pyruvate and 2-oxoglutarate are established specific inhibitors of cognate 2-oxo acid dehydrogenases. The present work develops application of this class of compounds to specific in vivo inhibition of 2-oxoglutarate dehydrogenase (OGDH) and its isoenzyme, 2-oxoadipate dehydrogenase (OADH). The isoenzymes-enriched preparations from the rat tissues with different expression of OADH and OGDH are used to characterize their interaction with 2-oxoglutarate (OG), 2-oxoadipate (OA) and the phosphonate analogs. Despite a 100-fold difference in the isoenzymes ratio in the heart and liver, similar Michaelis saturations by OG are inherent in the enzyme preparations from these tissues ( K m O G = 0.45 ± 0.06 and 0.27 ± 0.026 mM, respectively), indicating no significant contribution of OADH to the OGDH reaction, or similar affinities of the isoenzymes to OG. However, the preparations differ in the catalysis of OADH reaction. The heart preparation, where OADH/OGDH ratio is ≈ 0.01, possesses low-affinity sites to OA ( K m O A = 0.55 ± 0.07 mM). The liver preparation, where OADH/OGDH ratio is ≈ 1.6, demonstrates a biphasic saturation with OA: the low-affinity sites ( K m , 2 O A = 0.45 ± 0.12 mM) are similar to those of the heart preparation; the high-affinity sites ( K m , 1 O A = 0.008 ± 0.001 mM), revealed in the liver preparation only, are attributed to OADH. Phosphonate analogs of C5-C7 dicarboxylic 2-oxo acids inhibit OGDH and OADH competitively to 2-oxo substrates in all sites. The high-affinity sites for OA are affected the least by the C5 analog (succinyl phosphonate) and the most by the C7 one (adipoyl phosphonate). The opposite reactivity is inherent in both the low-affinity OA-binding sites and OG-binding sites. The C6 analog (glutaryl phosphonate) does not exhibit a significant preference to either OADH or OGDH. Structural analysis of the phosphonates binding to OADH and OGDH reveals the substitution of a tyrosine residue in OGDH for a serine residue in OADH among structural determinants of the preferential binding of the bulkier ligands to OADH. The consistent kinetic and structural results expose adipoyl phosphonate as a valuable pharmacological tool for specific in vivo inhibition of the DHTKD1-encoded OADH, a new member of mammalian family of 2-oxo acid dehydrogenases, up-regulated in some cancers and associated with diabetes and obesity.

Genome-Wide Transcriptome Profiling Provides Insight on Cholesterol and Lithocholate Degradation Mechanisms in Nocardioides simplex VKM Ac-2033D.

Steroid microbial degradation plays a significant ecological role for biomass decomposition and removal/detoxification of steroid pollutants. In this study, the initial steps of cholesterol degradation and lithocholate bioconversion by a strain with enhanced 3-ketosteroid dehydrogenase (3-KSD) activity, Nocardioides simplex VKM Ac-2033D, were studied. Biochemical, transcriptomic, and bioinformatic approaches were used. Among the intermediates of sterol sidechain oxidation cholest-5-en-26-oic acid and 3-oxo-cholesta-1,4-dien-26-oic acid were identified as those that have not been earlier reported for N. simplex and related species. The transcriptomic approach revealed candidate genes of cholesterol and lithocholic acid (LCA) catabolism by the strain. A separate set of genes combined in cluster and additional 3-ketosteroid Δ1-dehydrogenase and 3-ketosteroid 9α-hydroxylases that might be involved in LCA catabolism were predicted. Bioinformatic calculations based on transcriptomic data showed the existence of a previously unknown transcription factor, which regulates cholate catabolism gene orthologs. The results contribute to the knowledge on diversity of steroid catabolism regulation in actinobacteria and might be used at the engineering of microbial catalysts for ecological and industrial biotechnology.

Synthetic analogues of 2-oxo acids discriminate metabolic contribution of the 2-oxoglutarate and 2-oxoadipate dehydrogenases in mammalian cells and tissues.

The biological significance of the DHTKD1-encoded 2-oxoadipate dehydrogenase (OADH) remains obscure due to its catalytic redundancy with the ubiquitous OGDH-encoded 2-oxoglutarate dehydrogenase (OGDH). In this work, metabolic contributions of OADH and OGDH are discriminated by exposure of cells/tissues with different DHTKD1 expression to the synthesized phosphonate analogues of homologous 2-oxodicarboxylates. The saccharopine pathway intermediates and phosphorylated sugars are abundant when cellular expressions of DHTKD1 and OGDH are comparable, while nicotinate and non-phosphorylated sugars are when DHTKD1 expression is order(s) of magnitude lower than that of OGDH. Using succinyl, glutaryl and adipoyl phosphonates on the enzyme preparations from tissues with varied DHTKD1 expression reveals the contributions of OADH and OGDH to oxidation of 2-oxoadipate and 2-oxoglutarate in vitro. In the phosphonates-treated cells with the high and low DHTKD1 expression, adipate or glutarate, correspondingly, are the most affected metabolites. The marker of fatty acid ß-oxidation, adipate, is mostly decreased by the shorter, OGDH-preferring, phosphonate, in agreement with the known OGDH dependence of ß-oxidation. The longest, OADH-preferring, phosphonate mostly affects the glutarate level. Coupled decreases in sugars and nicotinate upon the OADH inhibition link the perturbation in glucose homeostasis, known in OADH mutants, to the nicotinate-dependent NAD metabolism.

Bioconversion of 6-(N-methyl-N-phenyl)aminomethyl androstane steroids by Nocardioides simplex.

The newly synthesized (α/ß)-diastereomers of 6-(N-methyl-N-phenyl)aminomethylandrost-4-ene-3,17-dione (5) and 6-(N-methyl-N-phenyl)aminomethylandrost-4-en-17ß-ol-3-one (6) were firstly investigated as substrates for the whole cells of Nocardioides simplex VKM Ac-2033D in comparison with their unsubstituted analogs, - androst-4-ene-3,17-dione (1) and androst-4-en-17ß-ol-3-one (2). 1(2)-Dehydroderivatives were identified as the major bioconversion products from all the substrates tested. When using the mixtures of (α/ß)-stereoisomers of 5 and 6 as the substrates, only ß-stereoisomers of the corresponding 1,4-diene-steroids were formed. Along with 1(2)-dehydrogenation, N. simplex VKM Ac-2033D promoted oxidation of the hydroxyl group at C-17 position of 6: both 6(α) and 6(ß) were transformed to the corresponding 17-keto derivatives. No steroid core destruction was observed during the conversion of the 6-substituted androstanes 5 and 6, while it was significant when 1 or 2 was used as the substrate. The results suggested high potentials of N. simplex VKM Ac-2033D for the generation of novel 1(2)-dehydroanalogs.

Specific inhibition by synthetic analogs of pyruvate reveals that the pyruvate dehydrogenase reaction is essential for metabolism and viability of glioblastoma cells.

The pyruvate dehydrogenase complex (PDHC) and its phosphorylation are considered essential for oncotransformation, but it is unclear whether cancer cells require PDHC to be functional or silenced. We used specific inhibition of PDHC by synthetic structural analogs of pyruvate to resolve this question. With isolated and intramitochondrial PDHC, acetyl phosphinate (AcPH, KiAcPH = 0.1 µM) was a much more potent competitive inhibitor than the methyl ester of acetyl phosphonate (AcPMe, KiAcPMe = 40 µM). When preincubated with the complex, AcPH also irreversibly inactivated PDHC. Pyruvate prevented, but did not reverse the inactivation. The pyruvate analogs did not significantly inhibit other 2-oxo acid dehydrogenases. Different cell lines were exposed to the inhibitors and a membrane-permeable precursor of AcPMe, dimethyl acetyl phosphonate, which did not inhibit isolated PDHC. Using an ATP-based assay, dependence of cellular viability on the concentration of the pyruvate analogs was followed. The highest toxicity of the membrane-permeable precursor suggested that the cellular action of charged AcPH and AcPMe requires monocarboxylate transporters. The relevant cell-specific transcripts extracted from Gene Expression Omnibus database indicated that cell lines with higher expression of monocarboxylate transporters and PDHC components were more sensitive to the PDHC inhibitors. Prior to a detectable antiproliferative action, AcPH significantly changed metabolic profiles of the investigated glioblastoma cell lines. We conclude that catalytic transformation of pyruvate by pyruvate dehydrogenase is essential for the metabolism and viability of glioblastoma cell lines, although metabolic heterogeneity causes different cellular sensitivities and/or abilities to cope with PDHC inhibition.

Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays.

Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay.