Aminotransferase Reaction Research Articles

Aspartate aminotransferase of Rhizobium leguminosarum has extended substrate specificity and metabolizes aspartate to enable N2 fixation in pea nodules.

Rhizobium leguminosarum aspartate aminotransferase (AatA) mutants show drastically reduced symbiotic nitrogen fixation in legume nodules. Whilst AatA reversibly transaminates the two major amino-donor compounds aspartate and glutamate, the reason for the lack of N2 fixation in the mutant has remained unclear. During our investigations into the role of AatA, we found that it catalyses an additional transamination reaction between aspartate and pyruvate, forming alanine. This secondary reaction runs at around 60 % of the canonical aspartate transaminase reaction rate and connects alanine biosynthesis to glutamate via aspartate. This may explain the lack of any glutamate-pyruvate transaminase activity in R. leguminosarum, which is common in eukaryotic and many prokaryotic genomes. However, the aspartate-to-pyruvate transaminase reaction is not needed for N2 fixation in legume nodules. Consequently, we show that aspartate degradation is required for N2 fixation, rather than biosynthetic transamination to form an amino acid. Hence, the enzyme aspartase, which catalyses the breakdown of aspartate to fumarate and ammonia, suppressed an AatA mutant and restored N2 fixation in pea nodules.

Considerations for the Scale‐up of in vitro Transaminase‐Catalyzed Asymmetric Synthesis of Chiral Amines

AbstractIn vitro transaminase catalyzed asymmetric synthesis is not a new phenomenon in the biocatalytic toolbox. Our group published a review on process consideration for these reactions in 2011, in which process metrics and the unfavorable reaction equilibrium was deduced to be the main bottlenecks at the time. Now, a decade later, this has been solved by different process methods presented in this review, with the development of enzymatic cascades one of the main solutions. Today, the new bottleneck is the downstream processing, due to the complexity of transaminase reactions, in which a ketone and an amine as substrates and a new ketone and a new amine as products. How to recover the produced amine from the resulting product mixture is considered and will be the key challenge for scale‐up of such systems in the near future.

Multi-substrate Metabolic Tracing Reveals Marked Heterogeneity and Dependency on Fatty Acid Metabolism in Human Prostate Cancer.

Prostate cancer utilizes multiple substrates to fuel energy requirements, yet pharmacologic targeting of fatty acid uptake and oxidation reveals metabolic dependencies in localized and metastatic tumors.

On the flexibility of the cellular amination network in E coli.

Ammonium (NH4+) is essential to generate the nitrogenous building blocks of life. It gets assimilated via the canonical biosynthetic routes to glutamate and is further distributed throughout metabolism via a network of transaminases. To study the flexibility of this network, we constructed an Escherichia coli glutamate auxotrophic strain. This strain allowed us to systematically study which amino acids serve as amine sources. We found that several amino acids complemented the auxotrophy either by producing glutamate via transamination reactions or by their conversion to glutamate. In this network, we identified aspartate transaminase AspC as a major connector between many amino acids and glutamate. Additionally, we extended the transaminase network by the amino acids β-alanine, alanine, glycine, and serine as new amine sources and identified d-amino acid dehydrogenase (DadA) as an intracellular amino acid sink removing substrates from transaminase reactions. Finally, ammonium assimilation routes producing aspartate or leucine were introduced. Our study reveals the high flexibility of the cellular amination network, both in terms of transaminase promiscuity and adaptability to new connections and ammonium entry points.

A phase II study evaluating KN026 monotherapy in patients (pts) with previously treated, advanced HER2-expressing gastric or gastroesophageal junction cancers (GC/GEJC).

4040 Background: Outcomes of second or later-line treatment for pts with advanced GC/GEJC remain inferior. KN026 is a novel HER2-targeted bispecific antibody composed of VH regions of trastuzumab and pertuzumab, targeting the HER2 juxtamembrane domain (IV) and the dimerization domain (II) simultaneously. KN026 has shown promising antitumor efficacy in preclinical and phase I studies. Here we present the results of KN026 monotherapy in previously treated, advanced HER2-expressing GC/GEJC. Methods: In this multi-center, single-arm, open-label, 2-cohort phase II study, adult pts with previously treated, advanced GC/GEJC were assigned into a HER2 high-level cohort (Cohort 1: IHC3+ or IHC 2+ ISH+) or a HER2 low-level cohort (Cohort 2: IHC 1+/2+ ISH- or IHC 0/1+ISH+). KN026 was given intravenously in 10 mg/kg QW, 20 mg/kg Q2W, or 30 mg/kg Q3W. Primary endpoints were objective response rate (ORR) and duration of response (DoR) assessed by investigators per RECIST 1.1. Secondary endpoints included progression-free survival (PFS), overall survival (OS), and safety outcomes. This trial is registered at ClinicalTrials.gov (NCT03925974). Results: At data cut-off (Oct. 29, 2021), 45 pts were enrolled and treated with KN026; 39 were eligible for response evaluation (25 pts in Cohort 1 and 14 pts in Cohort 2). In Cohort 1, ORR was 56% (95%CI 35%-76%) with 14 PR; the median DoR was 9.7 months (mo) (95%CI 4.2-not evaluable (NE)). At a median follow-up of 14.7 mo (95%CI 9.4-16.5), the median PFS was 8.3 mo (95%CI 4.2-11.4), and median OS was 16.3 mo (95% CI 11.0- NE). In Cohort 2, ORR was 14% (95%CI 2%-43%), with median DoR being 6.2 mo (95%CI 3.2-NE). At a median follow-up of 27.5 mo (95%CI 4.1-NE), the median PFS was 1.4 mo (95%CI 1.4-4.1), and median OS was 9.6 mo (95% CI 3.5-14.9). The table shows efficacy in Cohort 1 pts who have progressed on the prior trastuzumab treatment. KN026-related adverse events (TRAEs) were observed in 37 (82%) pts; the most common TRAEs (any grade) were increased aspartate aminotransferase (12, 27%), increased alanine aminotransferase (9, 20%), rash (7,16%), anemia (7,16%), and infusion-related reaction (7,16%). Four pts reported 5 grade 3 TRAEs, including infusion-related reaction, renal hydrocele, ureteral stenosis, increased blood pressure, and abnormal hepatic function (1 each). No grade 4 or 5 TRAEs occurred. Conclusions: KN026 monotherapy yielded promising efficacy with mild to moderate toxicities in pts with previously treated, advanced GC/GEJC. Further investigation is warranted. Clinical trial information: NCT03925974. [Table: see text]

The conundrum in enzymatic reactions related to biosynthesis of d-amino acids in bacteria.

d-Amino acids (d-AAs) are key components of the peptidoglycan matrix in bacterial cells. Various bacterial species are known to produce d-AAs by using different enzymes, such as highly specific and broad-spectrum racemases. Miyamoto etal. studied the biosynthesis of d-glutamate in the hyperthermophile and anaerobic Gram-negative bacterium, Thermotoga maritima, which does not possess a broad-spectrum racemase. The investigated TM0831 enzyme catalyzes both a d-amino acid aminotransferase reaction producing d-glutamate and an amino acid racemase activity aimed at generating d-aspartate and d-glutamate from the corresponding l-enantiomers. TM0831 represents an example of natural molecular evolution process favoring the enzyme versatility. Comment on: https://doi.org/10.1111/febs.16452.

Abrogating GPT2 in triple-negative breast cancer inhibits tumor growth and promotes autophagy.

Uncontrolled proliferation and altered metabolic reprogramming are hallmarks of cancer. Active glycolysis and glutaminolysis are characteristic features of these hallmarks and required for tumorigenesis. A fine balance between cancer metabolism and autophagy is a prerequisite of homeostasis within cancer cells. Here we show that glutamate pyruvate transaminase 2 (GPT2), which serves as a pivot between glycolysis and glutaminolysis, is highly upregulated in aggressive breast cancers, particularly the triple-negative breast cancer subtype. Abrogation of this enzyme results in decreased tricarboxylic acid cycle intermediates, which promotes the rewiring of glucose carbon atoms and alterations in nutrient levels. Concordantly, loss of GPT2 results in an impairment of mechanistic target of rapamycin complex 1 activity as well as the induction of autophagy. Furthermore, in vivo xenograft studies have shown that autophagy induction correlates with decreased tumor growth and that markers of induced autophagy correlate with low GPT2 levels in patient samples. Taken together, these findings indicate that cancer cells have a close network between metabolic and nutrient sensing pathways necessary to sustain tumorigenesis and that aminotransferase reactions play an important role in maintaining this balance.

Theoretical Analysis of the Built-in Metabolic Pathway Effect on the Metabolism of Erythrocyte-Bioreactors That Neutralize Ammonium.

The limitations of the efficiency of ammonium-neutralizing erythrocyte-bioreactors based on glutamate dehydrogenase and alanine aminotransferase reactions were analyzed using a mathematical model. At low pyruvate concentrations in the external medium (below about 0.3 mM), the main limiting factor is the rate of pyruvate influx into the erythrocyte from the outside, and at higher concentrations, it is the disappearance of a steady state in glycolysis if the rate of ammonium processing is higher than the critical value (about 12 mM/h). This rate corresponds to different values of glutamate dehydrogenase activity at different concentrations of pyruvate in plasma. Oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) by glutamate dehydrogenase decreases the fraction of NADPH in the constant pool of nicotinamide adenine dinucleotide phosphates (NADP + NADPH). This, in turn, activates the pentose phosphate pathway, where NADP reduces to NADPH. Due to the increase in flux through the pentose phosphate pathway, stabilization of the ATP concentration becomes impossible; its value increases until almost the entire pool of adenylates transforms into the ATP form. As the pool of adenylates is constant, the ADP concentration decreases dramatically. This slows the pyruvate kinase reaction, leading to the disappearance of the steady state in glycolysis.

The Impact of Fasting on Major Metabolic Pathways of Macronutrients and Pharmacokinetics Steps of Drugs.

In this review, we have investigated how fasting promotes an adaptive cross-talk between different hormones and metabolic pathways to supply and meet the body's daily energy demands. We highlight in biochemical terms and mechanisms how fasting impacts four metabolic pathways-glycogenolysis, gluconeogenesis, amino acid oxidation, and fatty acid β-oxidation-that are actively engaged in the metabolism of carbohydrates, proteins, and lipids. Fasting results in reduced insulin secretion and increased glucagon and epinephrine release to prevent or stimulate metabolic reaction(s). Fasting stimulates glycogenolysis, amino acid and glucose oxidation, aminotransferase reactions in skeletal muscle, and promotes gluconeogenesis and urea production in the liver. In addition, fasting promotes gene expression of lipid metabolism in skeletal muscle, the synthesis of ketone bodies in the liver, and intracellular hormone-sensitive lipase activity in adipose tissue. Furthermore, the impact of fasting on reducing cellular damage by mitochondrial reactive oxygen species is discussed. Lastly, we briefly describe the impact of fasting on the four steps of pharmacokinetics-the absorption, distribution, metabolism, and excretion of a few select drugs-with an emphasis on the elimination of drugs related to the cytochrome-P450 family of enzymes.

Novel method for measuring aquatic bacterial productivity using D10-leucine based on protein synthesis rate

The most widely used method for measuring bacterial production is tritium-labeled leucine (3H-Leu). Although this method provides methodological simplicity and high sensitivity, the employment of radioactive isotopes is often restricted by regulations, particularly in field settings. In this study, we developed a non-radioactive method for measuring bacterial productivity based on the protein synthesis rate, using deuterium-labeled leucine ((CD3)2CDCD2CD(NH2)COOH; D10-Leu); the proposed method was then compared and verified with the3H-Leu method. The procedures of the proposed method are (1) incorporation of D10-Leu by bacteria, (2) acid hydrolysis (HCl) to amino acids and (3) quantification of D10-Leu (m/z142.10) by liquid chromatography mass spectrometry (LC-MS/MS). In the LC-MS/MS analysis, we detected a larger amount of D9-Leu (m/z141.10) and D8-Leu (m/z140.10) than that of D10-Leu, suggesting that incorporated D10-Leu was rapidly metabolized such as in deamination and aminotransferase reactions. The incorporation rates of D10-Leu, D10-Leu + D9-Leu (D10+D9-Leu) and D10-Leu + D9-Leu + D8-Leu (D10+D9+D8-Leu) were significantly positively correlated to that of3H-Leu, confirming the validity of the proposed method. Since D7-Leu (m/z139.10) could not be detected, the amount of exogenous leucine incorporated into protein can be accurately estimated through D10+D9+D8-Leu measurement. The new compound-based quantification method using stable isotope-labeled leucine can be a powerful tool to estimate pure protein synthesis rate for measuring bacterial production.

Why Are Branched-Chain Amino Acids Increased in Starvation and Diabetes?

Branched-chain amino acids (BCAAs; valine, leucine, and isoleucine) are increased in starvation and diabetes mellitus. However, the pathogenesis has not been explained. It has been shown that BCAA catabolism occurs mostly in muscles due to high activity of BCAA aminotransferase, which converts BCAA and α-ketoglutarate (α-KG) to branched-chain keto acids (BCKAs) and glutamate. The loss of α-KG from the citric cycle (cataplerosis) is attenuated by glutamate conversion to α-KG in alanine aminotransferase and aspartate aminotransferase reactions, in which glycolysis is the main source of amino group acceptors, pyruvate and oxaloacetate. Irreversible oxidation of BCKA by BCKA dehydrogenase is sensitive to BCKA supply, and ratios of NADH to NAD+ and acyl-CoA to CoA-SH. It is hypothesized that decreased glycolysis and increased fatty acid oxidation, characteristic features of starvation and diabetes, cause in muscles alterations resulting in increased BCAA levels. The main alterations include (i) impaired BCAA transamination due to decreased supply of amino groups acceptors (α-KG, pyruvate, and oxaloacetate) and (ii) inhibitory influence of NADH and acyl-CoAs produced in fatty acid oxidation on citric cycle and BCKA dehydrogenase. The studies supporting the hypothesis and pros and cons of elevated BCAA concentrations are discussed in the article.

Use of a suspension‐t‐o‐suspension reaction in a continuously operated transaminase reaction system

Use of a suspension‐t‐o‐suspension reaction in a continuously operated transaminase reaction system

On the thermodynamics of biocatalytic reactions with application of group-contribution correlation and prediction

A thermodynamic analysis is presented to correlate and predict reaction equilibrium properties for enzyme-catalyzed synthesis of valuable chemical compounds, such as small-molecule pharmaceuticals. Utilization of group-contribution methods is shown for obtaining reference-state ideal gas and standard-state solution Gibbs energies of formation of reactants and products, as well as for estimating solution nonidealities. Several transaminase reactions have been treated. These cases were previously shown not to be accurately predicted by estimates of Gibbs energies of formation from group contributions. This work achieves accurate descriptions of the data by regressing some unknown component values with a method that accounts for uncertainties in property and data uncertainties. The results and methodology can be applied to many biosystems, but having some reaction equilibrium measurements with uncertainties that involve the species of interest are essential for reliable results.

Asymmetric Construction of Alkaloids by Employing a Key ω-Transaminase Cascade.

An ω-transaminase-triggered intramolecular aza-Michael reaction has been employed for the preparation of cyclic β-enaminones in good yield and excellent enantio- and diastereoselectivity, starting from easily accessible prochiral ketoynones and commercially available enzymes. The powerful thermodynamic driving force associated with the spontaneous aza-Michael reaction effectively displaces the transaminase reaction equilibrium towards product formation, using only two equivalents of isopropylamine. To demonstrate the potential of this methodology, this biocatalytic aza-Michael step was combined with annulation chemistry, affording unique stereo-defined fused alkaloid architectures.

Development of a Corynebacterium glutamicum bio-factory for self-sufficient transaminase reactions

The development and application of a self-sufficient whole-cell system for transaminase biotransformations is described. The system relies on an engineered strain of Corynebacterium glutamicum that produces smart amine donors.

Pyridoxal 5'-phosphate dependent reactions: Analyzing the mechanism of aspartate aminotransferase.

Enzyme catalysis is the primary activity in energy and information metabolism and enzyme cofactors are key to the catalytic ability of most enzymes. Pyridoxal 5'-phosphate (PLP) cofactor, derived from Vitamin B6, is widely distributed in nature and has significant latitude in catalytic diversity. X-ray crystallography has revealed the structures of diverse PLP dependent enzymes from multiple families. But these structures are incomplete, lacking the positions of protons essential for understanding enzymatic mechanisms. Here, we review the diversity of PLP and discuss the use of neutron crystallography and joint X-ray/neutron refinement of Fold Type I aspartate aminotransferase to visualize the positions of protons in both the internal and external aldimine forms. Strategies used to prepare extremely large crystals required for neutron diffraction and the approach to data refinement including the PLP cofactor are discussed. The observed positions of protons, including one located in a previously unknown low-barrier hydrogen bond, have been used to create more accurate models for computational analysis. The results revealed a new mechanism for the transaminase reaction where hyperconjugation is key to reducing the energy barrier which finally provides a clear explanation of the Dunathan alignment.

High Levels of Glutaminase II Pathway Enzymes in Normal and Cancerous Prostate Suggest a Role in 'Glutamine Addiction'.

Many tumors readily convert l-glutamine to α-ketoglutarate. This conversion is almost invariably described as involving deamidation of l-glutamine to l-glutamate followed by a transaminase (or dehydrogenase) reaction. However, mammalian tissues possess another pathway for conversion of l-glutamine to α-ketoglutarate, namely the glutaminase II pathway: l-Glutamine is transaminated to α-ketoglutaramate, which is then deamidated to α-ketoglutarate by ω-amidase. Here we show that glutamine transaminase and ω-amidase specific activities are high in normal rat prostate. Immunohistochemical analyses revealed that glutamine transaminase K (GTK) and ω-amidase are present in normal and cancerous human prostate and that expression of these enzymes increases in parallel with aggressiveness of the cancer cells. Our findings suggest that the glutaminase II pathway is important in providing anaplerotic carbon to the tricarboxylic acid (TCA) cycle, closing the methionine salvage pathway, and in the provision of citrate carbon in normal and cancerous prostate. Finally, our data also suggest that selective inhibitors of GTK and/or ω-amidase may be clinically important for treatment of prostate cancer. In conclusion, the demonstration of a prominent glutaminase II pathway in prostate cancer cells and increased expression of the pathway with increasing aggressiveness of tumor cells provides a new perspective on ‘glutamine addiction’ in cancers.

Crystal Structure Of Photorespiratory Alanine:Glyoxylate Aminotransferase 1 (AGT1) From Arabidopsis thaliana.

Photorespiration is an energetically costly metabolic pathway for the recycling of phosphoglycolate produced by the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) to phosphoglycerate. Arabidopsis alanine:glyoxylate aminotransferase 1 (AGT1) is a peroxisomal aminotransferase with a central role in photorespiration. This enzyme catalyzes various aminotransferase reactions, including serine:glyoxylate, alanine:glyoxylate, and asparagine:glyoxylate transaminations. To better understand structural features that govern the specificity of this enzyme, its crystal structures in the native form (2.2-Å resolution) and in the presence of l-serine (2.1-Å resolution) were solved. The structures confirm that this enzyme is dimeric, in agreement with studies of the active enzyme in solution. In the crystal, another dimer related by noncrystallographic symmetry makes close interactions to form a tetramer mediated in part by an extra carboxyl-terminal helix conserved in plant homologs of AGT1. Pyridoxal 5′-phosphate (PLP) is bound at the active site but is not held in place by covalent interactions. Residues Tyr35′ and Arg36′, entering the active site from the other subunits in the dimer, mediate interactions between AGT and l-serine when used as a substrate. In comparison, AGT1 from humans and AGT1 from Anabaena lack these two residues and instead position a tyrosine ring into the binding site, which accounts for their preference for l-alanine instead of l-serine. The structure also rationalizes the phenotype of the sat mutant, Pro251 to Leu, which likely affects the dimer interface near the catalytic site. This structural model of AGT1 provides valuable new information about this protein that may enable improvements to the efficiency of photorespiration.

Artificial Biocatalytic Cascade with Three Enzymes in One Pot for Asymmetric Synthesis of Chiral Unnatural Amino Acids

Two biocatalytic reactions, transamination catalyzed by transaminases and reductive amination catalyzed by amino acid dehydrogenases, can be used for asymmetric synthesis of optically pure unnatural amino acids. However, although transaminases show a great diversity and broad substrate spectrum, most transaminase reactions are reversible, while amino acid dehydrogenases catalyze reductive amination irreversibly but with strict substrate specificity. Accordingly, herein we developed a tri‐enzyme one‐pot reaction system to exploit the respective advantages of transaminases and amino acid dehydrogenases, while overcoming the disadvantages of each. In this work, representatives of all four subgroups of transaminases coupled with different amino acid dehydrogenases to produce five l‐ and four d‐ unnatural amino acid products, using ammonia and the co‐enzyme NAD(P)H, which is regenerated by a robust alcohol dehydrogenase with 2‐propanol as cheap cosubstrate. The complete conversion and high enantiopurity (ee > 99 %) of the products, demonstrated it as an ideal alternative for asymmetric synthesis of chiral amino acid compounds.

Application of leucine dehydrogenase Bcd from Bacillus subtilis for l-valine synthesis in Escherichia coli under microaerobic conditions

Microaerobic cultivation conditions have been shown experimentally and theoretically to improve the performance of a number of bioproduction systems. However, under these conditions, the production of l-valine by Escherichia coli is decreased mainly because of a redox cofactor imbalance and a decreased l-glutamate supply. The synthesis of one mole of l-valine from one mole of glucose generates two moles of NADH via glycolysis but consumes a total of two moles of NADPH, one in the ketol-acid reductoisomerase (KARI) reaction and the other in the regeneration of l-glutamate as an amino group donor for the branched-chain amino acid aminotransferase (BCAT) reaction. The improvement of l-valine synthesis under oxygen deprivation may be due to solving these problems. Increased l-valine synthesis under oxygen deprivation conditions was previously shown in Corynebacterium glutamicum (Hasegawa et al., 2012). In this study, we have proposed the use of NADH-dependent leucine dehydrogenase (LeuDH; EC 1.4.1.9) Bcd from B. subtilis instead of the native NADPH-dependent pathway including aminotransferase encoded by ilvE to improve l-valine production in E. coli under microaerobic conditions. We have created l-valine-producing strains on the base of the aminotransferase B-deficient strain V1 (B-7 ΔilvBN ΔilvIH ΔilvGME::PL-ilvBNN17KDA) by introducing one chromosomal copy of the bcd gene or the ilvE gene. Evaluation of the l-valine production by the obtained strains under microaerobic and aerobic conditions revealed that leucine dehydrogenase Bcd had a higher potential for l-valine production under microaerobic conditions. The Bcd-possessing strain exhibited 2.2-fold higher l-valine accumulation (up to 9.1 g/L) and 2.0-fold higher yield (up to 35.3%) under microaerobic conditions than the IlvE-possessing strain. The obtained results could be interpreted as follows: an altering of redox cofactor balance in the l-valine biosynthesis pathway increased the production and yield by E. coli cells under microaerobic conditions. Thus, the effective synthesis of l-valine by means of “valine fermentation” was shown in E. coli. This methodology has the advantages of being an economical and environmentally friendly process.