In vitro metabolic engineering for the production of α-ketoglutarate

α-Ketoglutarate (aKG) represents a central intermediate of cell metabolism. It is used for medical treatments and as a chemical building block. Enzymatic cascade reactions have the potential to sustainably synthesize this natural product. Here we report a systems biocatalysis approach for an in vitro reaction set-up to produce aKG from glucuronate using the oxidative pathway of uronic acids. Because of two dehydrations, a decarboxylation, and reaction conditions favoring oxidation, the pathway is driven thermodynamically towards complete product formation. The five enzymes (including one for cofactor recycling) were first investigated individually to define optimal reaction conditions for the cascade reaction. Then, the kinetic parameters were determined under these conditions and the inhibitory effects of substrate, intermediates, and product were evaluated. As cofactor supply is critical for the cascade reaction, various set-ups were tested: increasing concentrations of the recycling enzyme, different initial NAD⁺ concentrations, as well as the use of a bubble reactor for faster oxygen diffusion. Finally, we were able to convert 10 g L⁻¹ glucuronate with 92% yield of aKG within 5 h. The maximum productivity of 2.8 g L⁻¹ h⁻¹ is the second highest reported in the biotechnological synthesis of aKG.     

Inhibition by N′-nitrosonornicotine of the catalytic activity of glutamate dehydrogenase in α-ketoglutarate amination

The effect of N′-nitrosonornicotine (NNN), one of the tobacco-specific nitrosamines, on the catalytic activity of glutamate dehydrogenase (GLDH) in the α-ketoglutarate amination, using reduced nicotinamide adenine dinucleotide as coenzyme, was studied by a chronoamperometric method. The maximum reaction rate of the enzyme-catalyzed reaction and the Michaelis-Menten constant, or the apparent Michaelis-Menten constant, were determined in the absence and presence of NNN. NNN remarkably inhibited the bio-catalysis activity of GLDH, and was a reversible competitive inhibitior with K_i, estimated as 199?μmol?l^?1 at 25°C and pH 8.0.     

Glutamine and α-ketoglutarate as glutamate sources for glutathione synthesis in human erythrocytes

Glutathione (GSH) is an intracellular antioxidant synthesized from glutamate, cysteine and glycine. The human erythrocyte (red blood cell, RBC) requires a continuous supply of glutamate to prevent the limitation of GSH synthesis in the presence of sufficient cysteine, but the RBC membrane is almost impermeable to glutamate. As optimal GSH synthesis is important in diseases associated with oxidative stress, we compared the rate of synthesis using two potential glutamate substrates, α-ketoglutarate and glutamine. Both substrates traverse the RBC membrane rapidly relative to many other metabolites. In whole RBCs partially depleted of intracellular GSH and glutamate, 10 mm extracellular α-ketoglutarate, but not 10 mm glutamine, significantly increased the rate of GSH synthesis (0.85 ± 0.09 and 0.61 ± 0.18 μmol·(L RBC)(-1) ·min(-1), respectively) compared with 0.52 ± 0.09 μmol·(L RBC)(-1) ·min(-1) for RBCs without an external glutamate source. Mathematical modelling of the situation with 0.8 mm extracellular glutamine returned a rate of glutamate production of 0.36 μmol·(L RBC)(-1) ·min(-1), while the initial rate for 0.8 mM α-ketoglutarate was 0.97 μmol·(L RBC)(-1) ·min(-1). However, with normal plasma concentrations, the calculated rate of GSH synthesis was higher with glutamine than with α-ketoglutarate (0.31 and 0.25?μmol·(L RBC)(-1) ·min(-1), respectively), due to the substantially higher plasma concentration of glutamine. Thus, a potential protocol to maximize the rate of GSH synthesis would be to administer a cysteine precursor plus a source of α-ketoglutarate and/or glutamine.     

Disruption of genes for the enhanced biosynthesis of α-ketoglutarate in Corynebacterium glutamicum

The development of microbial strains for the enhanced production of α-ketoglutarate (α-KG) was investigated using a strain of Corynebacterium glutamicum that overproduces of l-glutamate, by disrupting three genes involved in the α-KG biosynthetic pathway. The pathways competing with the biosynthesis of α-KG were blocked by knocking out aceA (encoding isocitrate lyase, ICL), gdh (encoding glutamate dehydrogenase, l-gluDH), and gltB (encoding glutamate synthase or glutamate-2-oxoglutarate aminotransferase, GOGAT). The strain with aceA, gltB, and gdh disrupted showed reduced ICL activity and no GOGAT and l-gluDH activities, resulting in up to 16-fold more α-KG production than the control strain in flask culture. These results suggest that l-gluDH is the key enzyme in the conversion of α-KG to l-glutamate; therefore, prevention of this step could promote α-KG accumulation. The inactivation of ICL leads the carbon flow to α-KG by blocking the glyoxylate pathway. However, the disruption of gltB did not affect the biosynthesis of α-KG. Our results can be applied in the industrial production of α-KG by using C. glutamicum as producer.     

Effect of aminooxyacetate and α-ketoglutarate on glutamate deamination by rat kidney mitochondria

• 1.1. Glutamate dehydrogenase flux by rat kidney mitochondria incubated with 1 mM glutamine plus 2–3 mM glutamate was stimulated by aminooxyacetate. This effect was inhibited by α-ketoglutarate.
• 2.2. Studies with intact mitochondria and mitochondrial sonicates revealed a linear inverse relationship between glutamate deamination and α-ketoglutarate levels.
• 3.3. The data revealed that α-ketoglutarate is a competitive inhibitor of glutamate dehydrogenase with an apparent K_i of 0.6mM.
• 4.4. The data suggest that aminooxyacetate stimulates glutamate deamination by a mechanism mediated by α-ketoglutarate.

     

Separation and purification of α-ketoglutarate and pyruvate from the fermentation broth of Yarrowia lipolytica

Bioprocess and Biosystems Engineering - A strategy to achieve the efficient co-production of α-ketoglutarate (KGA) and pyruvate (PYR) via Yarrowia lipolytica fermentation was established in...     

Inactivation and reactivation of the mitochondrial α-ketoglutarate dehydrogenase complex

Reduced brain metabolism is an invariant feature of Alzheimer Disease (AD) that is highly correlated to the decline in brain functions. Decreased activities of key tricarboxylic acid cycle (TCA) cycle enzymes may underlie this abnormality and are highly correlated to the clinical state of the patient. The activity of the α-ketoglutarate dehydrogenase complex (KGDHC), an arguably rate-limiting enzyme of the TCA cycle, declines with AD, but the mechanism of inactivation and whether it can be reversed remains unknown. KGDHC consists of multiple copies of three subunits. KGDHC is sensitive to oxidative stress, which is pervasive in AD brain. The present studies tested the mechanism for the peroxynitrite-induced inactivation and subsequent reactivation of purified and cellular KGDHC. Peroxynitrite inhibited purified KGDHC activity in a dose-dependent manner and reduced subunit immunoreactivity and increased nitrotyrosine immunoreactivity. Nano-LC-MS/MS showed that the inactivation was related to nitration of specific tyrosine residues in the three subunits. GSH diminished the nitrotyrosine immunoreactivity of peroxynitrite-treated KGDHC, restored the activity and the immunoreactivity for KGDHC. Nano-LC-MS/MS showed this was related to de-nitration of specific tyrosine residues, suggesting KGDHC may have a denitrase activity. Treatment of N2a cells with peroxynitrite for 5 min followed by recovery of cells for 24 h reduced KGDHC activity and increased nitrotyrosine immunoreactivity. Increasing cellular GSH in peroxynitrite-treated cells rescued KGDHC activity to the control level. The results suggest that restoring KGDHC activity is possible and may be a useful therapeutic approach in neurodegenerative diseases.     

Solid state fermentation for the production of α-amylase from Penicillium chrysogenum using mixed agricultural by-products as substrate

Production of α-amylase from local isolate, Penicillium chrysogenum, under solid-state fermentation (SSF) was carried out in this study. Different agricultural by-products, such as wheat bran (WB), sunflower oil meal (SOM), and sugar beet oil cake (SBOC), were used as individual substrate for the enzyme production. WB showed the highest enzyme activity (750 U/gds). Combination of WB, SOM, and SBOC (1:3:1 w/w/w) resulted in a higher enzyme yield (845 U/gds) in comparison with the use of the individual substrate. This combination was used as mixed solid substrate for the production of α-amylase from P. chrysogenum by SSF. Fermentation conditions were optimized. Maximum enzyme yield (891 U/gds) was obtained when SSF was carried out using WB + SOM + SBOC (1:3:1 w/w/w), having initial moisture of 75%, inoculum level of 20%, incubation period of 7 days at 30°C. Galactose (1% w/w), urea and peptone (1% w/w), as additives, caused increase in the enzyme activity.     

The ruminant parasite Haemonchus contortus expresses an α1,3-fucosyltransferase capable of synthesizing the Lewis x and sialyl Lewis x antigens

Glycoproteins from the ruminant helminthic parasite Haemonchus contortus react with Lotus tetragonolobus agglutinin and Wisteria floribunda agglutinin, which are plant lectins that recognize α1,3-fucosylated GlcNAc and terminal β-GalNAc residues, respectively. However, parasite glycoconjugates are not reactive with Ricinus communis agglutinin, which binds to terminal β-Gal, and the glycoconjugates lack the Lewis x (Lex) antigen or other related fucose-containing antigens, such as sialylated Lex, Lea, Leb Ley, or H-type 1. Direct assays of parasite extracts demonstrate the presence of an α1,3-fucosyltransferase (α1,3FT) and β1,4-N-acetylgalactosaminyltransferase (β1,4GalNAcT), but not β1,4-galactosyltransferase. The H. contortus α1,3FT can fucosylate GlcNAc residues in both lacto-N-neotetraose (LNnT) Galα1→4GlcNAcβ1→3Galβ1→4Glc to form lacto-N-fucopentaose III Galβ1→ 4[Fucα1→3]GlcNAcβ1→3Galβ1→4Glc, which contains the Lex antigen, and the acceptor lacdiNAc (LDN) GalNAcβ1→4GlcNAc to form GalNAcβ1→4[Fucα1 →3]GlcNAc. The α1,3FT activity towards LNnT is dependent on time, protein, and GDP-Fuc concentration with a Km 50 μ M and a Vmax of 10.8 nmol-mg?1 h?1. The enzyme is unusually resistant to inhibition by the sulfhydryl-modifying reagent N-ethylmaleimide. The α1,3FT acts best with type-2 glycan acceptors (Galβ1→4GlcNAcβ1-R) and can use both sialylated and non-sialylated acceptors. Thus, although in vitro the H. contortus α1,3FT can synthesize the Lex antigen, in vivo the enzyme may instead participate in synthesis of fucosylated LDN or related structures, as found in other helminths.     

Experimental evidence that maleic acid markedly compromises glutamate oxidation through inhibition of glutamate dehydrogenase and α-ketoglutarate dehydrogenase activities in kidney of developing rats

Molecular and Cellular Biochemistry - This study was aimed to explore the role of C1q/TNF-related protein 9 (CTRP9) on atherosclerotic lesion formation. A recombinant lentiviral vector carrying...     

Kinetic properties and substrate inhibition of α-galactosidase from Aspergillus niger

The recombinant AglB produced by Pichia pastoris exhibited substrate inhibition behavior for the hydrolysis of p-nitrophenyl α-galactoside, whereas it hydrolyzed the natural substrates, including galactomanno-oligosaccharides and raffinose family oligosaccharides, according to the Michaelian kinetics. These contrasting kinetic behaviors can be attributed to the difference in the dissociation constant of second substrate from the enzyme and/or to the ability of the leaving group of the substrates. The enzyme displays the grater k_cat/K_m values for hydrolysis of the branched α-galactoside in galactomanno-oligosaccharides than that of raffinose and stachyose. A sequence comparison suggested that AglB had a shallow active-site pocket, and it can allow to hydrolyze the branched α-galactosides, but not linear raffinose family oligosaccharides.     

What is the blood concentration of extracellular vesicles? Implications for the use of extracellular vesicles as blood-borne biomarkers of cancer

Circulating biomarkers have a great potential in diagnosing cancer diseases at early stages, where curative treatment is a realistic possibility. In the recent years, using extracellular vesicles (EVs) derived from blood as biomarkers has gained widespread popularity, mainly because they are thought to be easy to isolate and carry a vast variety of biological cargos that can be analyzed for biomarker purposes. However, our current knowledge on the plasma EV concentration in normophysiological states is sparse. Here, we provide the very first mean estimate of the plasma EV concentration based on values obtained from a thorough literature review. The different estimates obtained from the literature are correlated to the isolation techniques used to obtain them, illustrating how some methodologies may over- or underestimate the plasma EV concentration. We also show that the estimated plasma EV concentration (approximately 10¹⁰ EVs per mL) defines EVs as a minority population compared to other colloidal particles of the systemic circulation, namely the lipoproteins, which are known contaminants in EV isolates and carry biomarker molecules themselves. Lastly, we introduce the possibility of regarding EVs and lipoproteins as a continuum of lipid-containing particles to which biomarker molecules can be associated. Using such a holistic approach, increased strength of plasma-derived cancer biomarkers may soon be revealed.     

Modifying the determinants of α-ketoacid substrate selectivity in mycobacterium tuberculosis α-isopropylmalate synthase

α-Isopropylmalate synthase (IPMS) catalyses the reaction between α-ketoisovalerate and acetyl coenzyme A (AcCoA) in the first step of leucine biosynthesis. IPMS is closely related to homocitrate synthase, which catalyses the reaction between AcCoA and the unbranched α-ketoacid α-ketoglutarate. Analysis of these enzymes suggests that several differently conserved key residues are responsible for the different substrate selectivity. These residues were systematically substituted in the Mycobacterium tuberculosis IPMS, resulting in changes in substrate specificity. A variant of IPMS was constructed with a preference for the unbranched α-ketoacids α-ketobutyrate and pyruvate over the natural branched substrate α-ketoisovalerate.     

Influence of the protein conformation on the interaction between α-lactalbumin and dimyristoylphosphatidylcholine vesicles

α-Lactalbumin is a globular protein containing helical regions with highly amphiphathic character. In this work, the interaction between bovine α-lactalbumin and sonicated dimyristoylphosphatidylcholine vesicles has been compared in different circumstances which influence the protein conformation i.e., pH, ionic strength, decalcification, guanidine hydrochloride denaturation. Above the isoelectric point the interaction is mainly electrostatic; improved electrostatic interaction results in better contact with the apolar lipid phase. Below the isoelectric point, hydrophobic forces dominate the interaction and the vesicles are solubilized. The mode of interaction is not determined to a great extent by the demetallization of the protein. However, by a more explicit unfolding of the globular structure with guanidine hydrochloride, micellar complexes can be formed with the lipid, even at neutral pH. From this study it is obvious that the presence or capability for formation of helices with high amphipathic character is not a sufficient condition for lipid solubilization by a globular protein. Also, the capability of a globular protein to unfold its tertiary structure seems to be a prerequisite for its capability to lipid solubilization.     

Specific inhibition of an α-galactosyltransferase from Trypanosoma brucei by synthetic substrate analogues

Since the alpha-D-galactose-(1-->3)-D-galactose epitope has been identified to be the major target in the process of hyperacute rejection of xenografts transplanted from nonprimate donors to humans, specific inhibitors of alpha-galactosyltransferases are of broad interest. Using Trypanosoma brucei, a protozoan parasite causing sleeping sickness and Nagana, we have a very useful model system for the investigation of alpha-galactosyltransferase inhibitors, since the variant surface glycoprotein (VSG) accounts for about 10% of the total cell protein an this parasite expresses many different galactosyltransferases including the one catalysing the formation of the Galalpha1-->3Gal epitope. In order to study inhibition of galactosylation on the VSG from Trypanosoma brucei, we designed, synthesized and tested substrate analogues of trypanosomal alpha-galactosyltransferases. Effective inhibitors were a pair of diastereoisomeric UDP-galactose analogs, in which the galactose residue is linked to UDP via a methylene bridge rather than an ester linkage. Hence, galactose cannot be transferred to the respective acceptor substrate VSG or the synthetic acceptor substrate Manalpha1-->6Manalpha1S-(CH2)7-CH3, which was previously proven to replace VSG effectively [Smith et al. (1996) J Biol Chem 271:6476-82]. Inhibitors have been prepared starting from 1-formyl galactal. The final condensation was performed using UMP morpholidate leading to a pair of diastereomeric compounds in 39% or 30% yield, respectively. These compounds were tested using alpha-galactosyltransferases prepared from T. brucei membranes and lactose synthetase from bovine milk. While the K(M)-value for UDP-galactose was determined as 59 microM on bovine lactose synthetase, the K(I)-values for both inhibitors were 0.3 mM and 1.1 mM respectively, showing that these inhibitors are unable to inhibit enzyme activity significantly. However, using the N-glycan specific alpha-galactosyltransferase from trypanosomes, the K(M)-value was determined as 20 microM, while the K(I)-values were 34 microM and 21 microM respectively. Interestingly, other trypanosomal alpha-galactosyltransferases, which modify the GPI membrane anchor, are 2 orders of magnitude less effected by the inhibitor.     

Enhanced production of α-ketoglutarate by fed-batch culture in the metabolically engineered strains of Corynebacterium glutamicum

The fed-batch culture system was employed to enhance production of α-ketoglutarate (α-KG) by the strainsof Corynebacterium glutamicum, whose genes encoding the key enzymes responsible for the biosynthesis of L-glutamate from α-KG were deleted. In a shake flask fermentation, C. glutamicum JH110 in which the 3 genes, gdh (encoding glutamate dehydrogenase), gltB (encoding glutamate synthase), and aceA (encoding isocitrate lyase) were disrupted showed the highest production of α-KG (12.4 g/L) compared to the strains JH102 (gdh mutant), JH103 (gltB mutant), and JH107 (gdh gltB double mutant). In the fed-batch cultures using a 5 L-jar fermenter, the strain JH107 produced more α-KG (19.5 g/L), but less glutamic acid (23.3 g/L) than those produced by the parent strain HH109, as well as JH102. The production of α-KG was significantly enhanced and the accumulation of glutamicacid was minimized by the ammonium-limited fed-batch cultures employing C. glutamicum JH107. Further improvement of α-KG production by the strain JH107 was achieved through the ammonium-limited fed-batch culture with the feeding of molasses, and the levels of α-KG and glutamic acid produced were 51.1 and 0.01 g/L, respectively.