Α-keto Acids Research Articles

Efficient whole cell biotransformation of tyrosol from L-tyrosine by engineered Escherichia coli

An engineered Escherichia coli was constructed by co-expressing L-amino acid deaminase, α-keto acid decarboxylase, alcohol dehydrogenase, and glucose dehydrogenase through two plasmids for tyrosol production. The activity of the rate-limiting enzyme L-amino acid deaminase from Cosenzaea myxofaciens (CmAAD) toward tyrosine was improved by structure-guided modification. The enzyme activity of triple mutant CmAAD V438G/K147V/R151E toward tyrosine was ~5.12-fold higher than that of the wild-type CmAAD. Secondly, the plasmid copy numbers and the gene orders were optimized to improve the titer of tyrosol. Finally, the recombinant strain CS-6 transformed 10 mM tyrosine into 9.56 ± 0.64 mM tyrosol at 45 ℃, and the space-time yield reached 0.478 mM·L−1·h−1. This study proposes a novel idea for the efficient and natural production of tyrosol, which has great potential for industrial application.

The Role of Branched-Chain Amino Acids and Branched-Chain α-Keto Acid Dehydrogenase Kinase in Metabolic Disorders.

Branched-chain amino acids (BCAAs), composed of leucine, isoleucine, and valine, are important essential amino acids in human physiology. Decades of studies have revealed their roles in protein synthesis, regulating neurotransmitter synthesis, and the mechanistic target of rapamycin (mTOR). BCAAs are found to be related to many metabolic disorders, such as insulin resistance, obesity, and heart failure. Also, many diseases are related to the alteration of the BCAA catabolism enzyme branched-chain α-keto acid dehydrogenase kinase (BCKDK), including maple syrup urine disease, human autism with epilepsy, and so on. In this review, diseases and the corresponding therapies are discussed after the introduction of the catabolism and detection methods of BCAAs and BCKDK. Also, the interaction between microbiota and BCAAs is highlighted.

Synthesis of Peptide α-Keto Acids and Application to Decarboxylative Peptide Coupling

Key words organocatalysis - Mannich reaction - peptide keto acids - peptide coupling - decarboxylation

P338: BRANCHED CHAIN AMINO ACID TRANSAMINASE 1 ASSOCIATES WITH THE KU70/KU86 HETERODIMER AND MODULATES THE DNA DAMAGE RESPONSE IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA CELLS

Background: T-cell acute lymphoblastic leukemia (T-ALL) is a biologically and clinically heterogeneous disease mostly associated with NOTCH1 mutations that characterize over 60% of cases. Therapy-resistant or refractory T-ALL remains a major clinical challenge. Branched Chain Amino Acid Transferase 1 (BCAT1), a cytosolic aminotransferase converts BCAAs into their corresponding branched-chain α-keto acids and vice versa. Aberrant expression of BCAT1 has been demonstrated in different tumor models, including acute leukemia. However, its biological role in T-ALL remains to be elucidated. Aims: The purpose of this study was to dissect the functional role of BCAT1 in T-ALL, with particular emphasis on a putative link between BCAT1 and NOTCH1 in promoting T-ALL initiation and progression. Methods: We evaluated BCAT1 expression using RNAseq data from 264 T-ALL samples (ALL0434 protocol). BCAT1 transcript and protein levels were determined in T-ALL cell lines (n=13) and patient derived xenografts (PDX; n=12). Metabolomics analysis (using 13C6 labeled Leucine) was performed following NOTCH1 inhibition. Further, five PDX samples were treated with a γ secretase inhibitor (DBZ) to dampen NOTCH signaling. BCAT1 promoter occupancy by NOTCH1 was determined by Chromatin Immunoprecipitation (ChIP) coupled with qPCR. Tandem affinity purification (TAP) and mass spectrometry (MS) analysis were used to identify BCAT1 interacting proteins from CUTLL1 T-ALL cells. Kinetics of the DNA damage response (DDR) was evaluated in BCAT1 knock-down cells. Drug combination experiments using the DNA damaging agent (etoposide) and a specific BCAT inhibitor (ERG245) were performed in vitro and in vivo in PDX T-ALL models. Results: We found variable BCAT1 expression levels amongst the different T-ALL molecular subgroups, with higher expression levels in TLX1 (p<0.001), HOXA-TLX3 (p<0.05) and NKX2-1 (p<0.01) subtypes. Considering recurrent genetic alterations, we found BCAT1 expression to be significantly higher (p<0.01) in NOTCH1 mutated cases compared to un-mutated samples. Interestingly, NOTCH1 mutated PDX samples also showed higher BCAT1 expression compared to un-mutated cases. Metabolic studies disclosed that BCAAs oxidation is decreased following NOTCH1 inhibition (reduction in 3-methyl-2 oxovaleric acid). Further, inhibition of NOTCH1 signaling with DBZ decreased BCAT1 expression in numerous T-ALL models. ChIP analysis and luciferase reporter assays suggest that NOTCH1 may directly regulate BCAT1 expression. Functionally, TAP followed by MS analysis of BCAT1 interacting proteins disclosed that BCAT1 may be implicated in non-metabolic processes such as DNA replication and repair and rRNA processing. Indeed, we found Ku70/Ku86 proteins to interact with BCAT1. Evaluation of the DDR following DNA damage, disclosed that BCAT1 deficient cells present an accentuated but defective DDR which translates into marked DNA damage leading to pronounced cell death. Consistently, a marked synergistic therapeutic response was found between the DNA damaging drug etoposide and the BCAT specific inhibitor ERG245 in numerous T-ALL models. Summary/Conclusion: NOTCH1 may directly regulate BCAT1 expression in T-ALL cells. BCAT1 is involved in sustaining genomic integrity following DNA damage through its interaction with the Ku70/Ku86 heterodimer, known to mediate classical non-homologous end-joining. Our results identify BCAT1 as a novel therapeutic target and suggest that the combination between DNA double stand break inducing agents (such as etoposide) and a BCAT inhibitor could be particularly useful in NOTCH1-mutant T-ALL cases.

191-OR: Hepatic Expression of the Branched-Chain Keto Acid Dehydrogenase Kinase Is Sufficient to Cause Glucose Intolerance in Lean Mice

Perturbed branched-chain amino acid homeostasis is a feature of obesity, insulin-resistance and type 2 diabetes. Induction of the branched-chain keto acid dehydrogenase kinase (BDK) in liver is one mechanism that is thought to contribute to elevated BCAA levels in these conditions. Whether higher levels of BDK in liver are sufficient to disturb systemic BCAA homeostasis and promote glucose intolerance in the absence of obesity or insulin resistance has never been directly tested. We treated 12 week-old C57BL6/J mice with adeno-associated virus, serotype 8 (AAV8) -BDK or control AAV8-GFP to promote long term expression of BDK specifically in liver of lean mice. Mice were then maintained on a standard low-fat chow diet for 8 weeks (n= 9 AAV8-GFP and n=AAV8-BDK) . AAV8-BDK significantly raised BDK protein and mRNA expression in liver but not adipose, skeletal muscle, or cardiac tissue compared to AAV8-GFP control mice. The higher expression of BDK in liver was associated with robust inhibitory phosphorylation of the branched-chain keto acid dehydrogenase (BCKDH) on ser293. AAV8-mediated induction of BDK expression in liver in our study was sufficient to promote significant elevations in circulating levels of the direct substrate of BCKDH, the branched chain α-keto acids as well as their cognate BCAA. There was no effect of AAV8-BDK to alter food intake, weight gain or adiposity. However, glucose tolerance measured by a 1g/kg intraperitoneal glucose tolerance test at week 7 was significantly impaired in mice receiving AAV8-BDK compared to AAV8-GFP control. These data demonstrate for the first time that induction of BDK in liver, as occurs with obesity, is sufficient to disturb systemic BCAA and glucose homeostasis in lean mice. Disclosure J.Walejko: None. R.W.Mcgarrah: None. P.J.White: None. Funding American Diabetes Association (1-16-INI-17)

Formation of Volatile Heterocyclic Compounds and Open-Chain Amides of Theanine in Model Systems with Glucose, Tea Leaves, and Tea Extract under Tea-Roasting Conditions.

Theanine is a non-proteinogenic amino acid found in the tea plant Camellia sinensis. At an elevated temperature (>90 °C), it released two major volatile compounds 1-ethyl-1,5-dihydro-2H-pyrrol-2-one and N-ethylsuccinimide. Other products were identified, including 10 pyrroles and 12 amides/imides. The formation of the two major compounds was proposed to be initiated by the deamination of theanine and through the intermediate α-keto acid. In the presence of glucose, the two major products and many other volatiles from theanine thermal degradation were accelerated and further Maillard reactions occurred. A total of 56 compounds were identified in the model system of theanine and glucose, including 12 amides/imides, 16 pyrazines, 16 pyrroles and other N-heterocycles, and 12 furans and other O-heterocycles. Although most of the reaction products were detected in tea leaves and in their aqueous extract with or without the addition of theanine under the same experiment conditions, imides and amides were considerably suppressed, left only minute amounts, or were even no longer detectable. Pyrazines and pyrroles were also shown at reduced concentrations as a result of the interaction with tea components but to a lesser extent. A total of 16 and 12 pyrazines were identified in the theanine/glucose reaction system and tea leaves/aqueous extract after roasting, respectively. The results indicated that pyrazines and other main volatiles in roasted tea leaves were formed from the Maillard reactions of the aqueous fraction of tea leaves. Theanine participated in the formation of pyrazines in tea leaves under roasting conditions.

MO980: Metabolomics Approach and Acute Rejection in Kidney Transplant Recipients

Abstract BACKGROUND AND AIMS Non-invasive diagnosis of graft rejection after kidney transplantation is still an unresolved issue. Changes in serum metabolite concentrations reflect the various processes involved in the graft, and thus graft rejection. Metabolomics approach might help with non-invasive diagnosis of graft rejection based on identification of T-cell activation. METHOD In a group of 55 patients after primary kidney transplantation (average follow-up 68.4 months, median 59 months) we identified patients with cellular rejection (ACR), antibody mediated rejection (AMR) and control group (CG)—patients without history of graft rejection. We used NMR metabolomic approach to determine the relative levels of metabolites in blood plasma. RESULTS From 23 evaluated metabolites: lactate was significantly increased in CG and in ACR against AMR (P &amp;lt; 0.05), whilst plasma glutamine decreased in CG against ACR (P &amp;lt; 0.05) and AMR (P &amp;lt; 0.05) groups. Further, tyrosine was significantly increased in CG against AMR (P &amp;lt; 0.05). All three branched chain α-keto acids (BCKAs) behaved similarly. However, little over significance cut off were CG &amp;gt;AMR. When discriminating CG and AMR, Random Forest discriminatory algorithm performed with AUC = 0.849 when included also evaluated plasma creatinine and with AUC = 0.83 when creatinine was excluded (other metabolites with the highest discriminatory power were glutamine, lactate, tyrosine and BCKAs). CONCLUSION Metabolomic approach to blood plasma in patients after kidney transplantation seems to be very promising when looking for non-invasive techniques assessing graft viability/survival. The observed metabolomic changes were able to very well discriminate between patients with AMR and control group. However, discrimination of ACR against CG and AMR was rather poor.

Alteration of the Catalytic Reaction Trajectory of a Vicinal Oxygen Chelate Enzyme by Directed Evolution

AbstractThe vicinal oxygen chelate (VOC) metalloenzyme superfamily catalyzes a highly diverse set of reactions with the mechanism characterized by the bidentate coordination of vicinal oxygen atoms to metal ion centers, but there remains a lack of a platform to steer the reaction trajectories, especially for o‐quinone metabolizing pathways. Herein, we present the directed‐evolution‐enabled bifunctional turnover of ChaP, which is a homotetramer and represents an unprecedented VOC enzyme class. Unlike the ChaP catalysis of extradiol‐like o‐quinone cleavage and concomitant α‐keto acid decarboxylation, a group of ChaP variants (CVs) catalyze intradiol‐like o‐quinone deconstruction and CO2 liberation from the resulting o‐hydroxybenzoic acid scaffolds with high regioselectivity. Enzyme crystal structures, labeling experiments and computational simulations corroborated that the D49L mutation allows the metal ion to change its coordination with the tyrosine phenoxy atoms in different monomers, thereby altering the reaction trajectory with the regiospecificity further improved by the follow‐up replacement of the Y92 residue with any of alanine, glycine, threonine, and serine. The study highlights the unpredicted catalytic versatility and enzymatic plasticity of VOC enzymes with biotechnological significance.

Alteration of the Catalytic Reaction Trajectory of a Vicinal Oxygen Chelate Enzyme by Directed Evolution.

The vicinal oxygen chelate (VOC) metalloenzyme superfamily catalyzes a highly diverse set of reactions with the mechanism characterized by the bidentate coordination of vicinal oxygen atoms to metal ion centers, but there remains a lack of a platform to steer the reaction trajectories, especially for o-quinone metabolizing pathways. Herein, we present the directed-evolution-enabled bifunctional turnover of ChaP, which is a homotetramer and represents an unprecedented VOC enzyme class. Unlike the ChaP catalysis of extradiol-like o-quinone cleavage and concomitant α-keto acid decarboxylation, a group of ChaP variants (CVs) catalyze intradiol-like o-quinone deconstruction and CO2 liberation from the resulting o-hydroxybenzoic acid scaffolds with high regioselectivity. Enzyme crystal structures, labeling experiments and computational simulations corroborated that the D49L mutation allows the metal ion to change its coordination with the tyrosine phenoxy atoms in different monomers, thereby altering the reaction trajectory with the regiospecificity further improved by the follow-up replacement of the Y92 residue with any of alanine, glycine, threonine, and serine. The study highlights the unpredicted catalytic versatility and enzymatic plasticity of VOC enzymes with biotechnological significance.

BCKDK Promotes Ovarian Cancer Proliferation and Migration by Activating the MEK/ERK Signaling Pathway.

Background Ovarian cancer (OC) is the most fatal gynecologic cancer. The branched-chain α-keto acid dehydrogenase kinase (BCKDK) plays an important role in many serious human diseases, including cancers. Its function in promoting cell proliferation and migration has been reported in various cancers. However, the biological role of BCKDK and its molecular mechanisms underlying OC initiation and progression are unclear. Methods First, the expression level of BCKDK in OC cell lines or tissues was determined using tissue microarray- (TMA-) based immunohistochemistry or western blotting. Then, growth curve analysis, anchorage-independent cell transformation assays, wound healing assays, cell migration assays, and tumor xenografts were used to test whether BCKDK could promote cell transformation or metastasis. Finally, the signaling pathways involved in this process were investigated by western blotting or immunoprecipitation. Results We found that the expression of BCKDK was upregulated in OC tissues and the high expression of BCKDK was correlated with an advanced pathological grade in patients. The ectopic overexpression of BCKDK promoted the proliferation and migration of OC cells, and the knockdown of BCKDK with shRNAs inhibited the proliferation and migration of OC ex vivo and in vivo. Moreover, BCKDK promoted OC proliferation and migration by activating MEK. Conclusions Our results demonstrate that BCKDK promotes OC proliferation and migration by activating the MEK/ERK signaling pathway. Targeting the BCKDK-MEK axis may provide a new therapeutic strategy for treating patients with OC.

Cloning, purification, and characterization of branched-chain α-keto acid decarboxylases from Lactococcus lactis strains with different 3-methylbutanal production abilities

Lactococcus lactis is an important food-grade microorganism that has been successfully applied as a starter to increase the level of 3-methylbutanal produced during the ripening of cheese. Three variants of branched-chain α-keto acid decarboxylase (KADC) were discovered in L. lactis strains with different 3-methylbutanal production abilities. Three genes encoding KADCs of varying lengths (KADC-long, KADC-middle, and KADC-short) were cloned and heterologously expressed into Escherichia coli. KADC activity was only detected in the E. coli cloned with the KADC-long-encoding gene. Homology modeling of the three KADC recombination proteins showed that an active-site residue (Glu462) and an S-pocket structure were necessary for the ability to catalyze substrates. KADC-long showed maximum activity at pH 7.0 and 30 °C. The substrate hydrolysis and kinetic parameters demonstrated that KADC-long efficiently produces 2-methylbutanal and 3-methylbutanal. The heterologous expression of the full-length kdcA in low-3-methylbutanal-yield L. lactis strains increased their production yields. The results of this study demonstrate the function of the complete KADC in 3-methylbutanal production.

Coadministration of tianeptine alters behavioral parameters and levels of neurotrophins in a chronic model of Maple Syrup Urine disease.

Maple Syrup Urine Disease (MSUD) is caused by the deficiency in the activity of the branched-chain α-ketoacid dehydrogenase complex (BCKDC), resulting in the accumulation of the branched-chain amino acids (BCAA) leucine, isoleucine, and valine, and their respective branched-chain α-keto acids. Patients with MSUD are at high risk of developing chronic neuropsychiatric disorders; however, the pathophysiology of brain damage in these patients remains unclear. We hypothesize that MSUD can cause depressive symptoms in patients. To test our hypothesis, Wistar rats were submitted to the BCAA and tianeptine (antidepressant) administration for 21days, starting seven days postnatal. Depression-like symptoms were assessed by testing for anhedonia and forced swimming after treatments. After the last test, the brain structures were dissected for the evaluation of neutrophins. We demonstrate that chronic BCAA administration induced depressive-like behavior, increased BDNF levels, and decreased NGF levels, suggesting a relationship between BCAA toxicity and brain damage, as observed in patients with MSUD. However, the administration of tianeptine was effective in preventing behavioral changes and restoring neurotrophins levels.

Coumarin derivatives as inhibitors of d-amino acid oxidase and monoamine oxidase

d-Amino acid oxidase (DAAO) oxidises d-amino acids to ultimately produce the corresponding α-keto acids. The DAAO substrate, d-serine, is a co-agonist at NMDA receptors, while NMDA receptor hypo-function has been implicated in the pathophysiology of schizophrenia. Through the modulation of d-serine levels, the inhibition of DAAO represents a strategy to increase NMDA receptor function, and thus a potential treatment for schizophrenia. Literature reports that 3-hydroxycoumarin is a potent inhibitor of DAAO and represents an ideal lead for the development of novel DAAO inhibitors. Based on this, the present study investigated DAAO inhibition by a series of synthetic and commercially available coumarin derivatives. Due to structural similarity to coumarin, a synthetic series of 3,4-dihydroisoquinolin-1(2H)-one derivatives has also been included in this study. The results show that among 37 compound evaluated, four inhibit porcine kidney DAAO with IC50 < 10 µM. The most potent inhibitors are 3,7-dihydroxycoumarin and 6,7-dihydroxycoumarin with an IC50 values of 0.167 and 0.224 µM, respectively. These values are an improvement on that of the reference DAAO inhibitor, 3-methylpyrazole-5-carboxylic acid (IC50 = 1.88 µM). Coumarin compounds are also known to inhibit the monoamine oxidase (MAO) enzymes, which are well established targets for the treatment of depression and Parkinson’s disease. As DAAO and MAO are flavoenzymes, off-target inhibition may occur. The series were thus evaluated as potential MAO inhibitors, and a number of high potency inhibitors were identified. Seven compounds inhibit the recombinant human MAOs with IC50 < 0.1 µM, with the most potent MAO-A and MAO-B inhibitors exhibiting IC50 values of 0.033 and 0.012 µM, respectively. This is significantly more potent than the reference inhibitors, curcumin, isatin and toloxatone. This study concludes that active DAAO and MAO inhibitors may serve as novel leads for the design of compounds that may find future application in the treatment of neuropsychiatric (e.g. schizophrenia, depression) and neurodegenerative disorders (e.g. Parkinson’s disease).

The Critical Role of the Branched Chain Amino Acids (BCAAs) Catabolism-Regulating Enzymes, Branched-Chain Aminotransferase (BCAT) and Branched-Chain α-Keto Acid Dehydrogenase (BCKD), in Human Pathophysiology.

Branched chain amino acids (BCAAs), leucine, isoleucine and valine, are essential amino acids widely studied for their crucial role in the regulation of protein synthesis mainly through the activation of the mTOR signaling pathway and their emerging recognition as players in the regulation of various physiological and metabolic processes, such as glucose homeostasis. BCAA supplementation is primarily used as a beneficial nutritional intervention in chronic liver and kidney disease as well as in muscle wasting disorders. However, downregulated/upregulated plasma BCAAs and their defective catabolism in various tissues, mainly due to altered enzymatic activity of the first two enzymes in their catabolic pathway, BCAA aminotransferase (BCAT) and branched-chain α-keto acid dehydrogenase (BCKD), have been investigated in many nutritional and disease states. The current review focused on the underlying mechanisms of altered BCAA catabolism and its contribution to the pathogenesis of a numerous pathological conditions such as diabetes, heart failure and cancer. In addition, we summarize findings that indicate that the recovery of the dysregulated BCAA catabolism may be associated with an improved outcome and the prevention of serious disease complications.

Determination of selected α-keto acids in dried blood samples using HPLC with fluorescence detection

The determination of α-keto acids derived from amino acids is currently the most reliable approach for the diagnosis of some congenital metabolic diseases. An HPLC method for the simultaneous measurement of selected α-keto acids in dried blood samples has been developed and evaluated. Blood spot samples from a group of healthy blood donors were collected onto #903 Specimen Collection Paper. Prior the separation, the α-keto acids were derivatized with 1,2-diamino-4,5-dimethoxybenzene to the corresponding 3-substituted-6,7-dimethoxy-2(1 H)-quinoxalinol derivatives. For the separation, a reverse-phase column LichroCart 125–4, Purospher RP-18e, 5 µm, was used. The mixture of 25% ACN in deionized water (mobile phase A) and 100% ACN (mobile phase B) were used for a gradient elution of α-keto acids derivatives. Analytical performance of this method is satisfactory for all α-keto acids. The intra-assay and inter-assay coefficients were below 10% and recoveries were close to 100%. We have developed relatively simple, rapid, selective and sufficiently sensitive HPLC method with fluorescence detection for the determination of selected α-keto acids in dried blood samples. The presented method is suitable for clinical testing purposes.

A Palladium-Catalyzed 4CzIPN-Mediated Decarboxylative Acylation Reaction of O-Methyl Ketoximes with α-Keto Acids under Visible Light.

This work presents a palladium-catalyzed oxime ether-directed ortho-selective benzoylation using benzoylformic acid as the acyl source with a palladium catalyst and 4CzIPN as the co-catalyst under light. Various non-symmetric benzophenone derivatives were obtained in moderate to good yields. A preliminary mechanism study revealed that the reaction proceeds through a free radical pathway.

Photoredox Neutral Decarboxylative Hydroxyalkylations of Heteroarenes with α-Keto Acids.

Photoredox neutral decarboxylative hydroxyalkylations of heteroarenes with α-keto acids under mild conditions are described. Stable and readily available α-keto acids were employed as hydroxyalkylating reagents with only CO2 released as the byproduct. A range of aromatic and aliphatic α-keto acids were successfully converted into hydroxyalkylated products with various heteroarenes. This transformation proceeded through a decarboxylation/Minisci addition/SCS sequence, generating a variety of valuable hydroxyalkylated heteroarenes.

Pyruvic acid, an efficient catalyst in SO&amp;lt;sub&amp;gt;3&amp;lt;/sub&amp;gt; hydrolysis and effective clustering agent in sulfuric-acid-based new particle formation

Abstract. The role of pyruvic acid (PA), one of the most abundant α-keto carboxylic acids in the atmosphere, was investigated both in the SO3 hydrolysis reaction to form sulfuric acid (SA) and in SA-based aerosol particle formation using quantum chemical calculations and a cluster dynamics model. We found that the PA-catalyzed SO3 hydrolysis is a thermodynamically driven transformation process, proceeding with a negative Gibbs free-energy barrier, ca. −1 kcal mol−1 at 298 K, ∼ 6.50 kcal mol−1 lower than that in the water-catalyzed SO3 hydrolysis. Results indicated that the PA-catalyzed reaction can potentially compete with the water-catalyzed SO3 reaction in SA production, especially in dry and polluted areas, where it is found to be ∼ 2 orders of magnitude more efficient that the water-catalyzed reaction. Given the effective stabilization of the PA-catalyzed SO3 hydrolysis product as SA⚫PA cluster, we proceeded to examine the PA clustering efficiency in a sulfuric-acid–pyruvic-acid–ammonia (SA-PA-NH3) system. Our thermodynamic data used in the Atmospheric Cluster Dynamics Code indicated that under relevant tropospheric temperatures and concentrations of SA (106 molec.cm-3), PA (1010 molec.cm-3) and NH3 (1011 and 5 × 1011 molec.cm-3), PA-enhanced particle formation involves clusters containing at most one PA molecule. Namely, under these monomer concentrations and 238 K, the (SA)2⚫PA⚫(NH3)2 cluster was found to contribute by ∼ 100 % to the net flux to aerosol particle formation. At higher temperatures (258 and 278 K), however, the net flux to the particle formation is dominated by pure SA-NH3 clusters, while PA would rather evaporate from the clusters at high temperatures and not contribute to the particle formation. The enhancing effect of PA was examined by evaluating the ratio of the ternary SA-PA-NH3 cluster formation rate to binary SA-NH3 cluster formation rate. Our results show that while the enhancement factor of PA to the particle formation rate is almost insensitive to investigated temperatures and concentrations, it can be as high as 4.7 × 102 at 238 K and [NH3] = 1.3 × 1011 molec.cm-3. This indicates that PA may actively participate in aerosol formation, only in cold regions of the troposphere and highly NH3-polluted environments. The inclusion of this mechanism in aerosol models may reduce uncertainties that prevail in modeling the aerosol impact on climate.

BDK knockout skeletal muscle satellite cells exhibit enhanced protein translation initiation signal in response to BCAA in vitro

We examined the effects of branched-chain amino acids (BCAAs) and electrical pulse stimulation (EPS) on the mTORC1 pathway in muscle satellite cells (MSCs) isolated from branched-chain α-keto acid dehydrogenase kinase (BDK) knockout (KO) mice in vitro. MSCs were isolated from BDK KO and wild-type (WT) mice, proliferated, and differentiated into myotubes. BCAA stimulation increased the phosphorylation of p70 S6 kinase (p70S6K), a marker of protein translation initiation, in MSCs from WT and BDK KO mice, but the rate of the increase was higher in MSCs isolated from BDK KO mice. Contrarily, there was no difference in the increase in p70S6K phosphorylation by EPS. Acute BDK knockdown in MSCs from WT mice using shRNA decreased p70S6K phosphorylation in response to BCAA stimulation. Collectively, the susceptibility of mTORC1 to BCAA stimulation was elevated by chronic, but not acute, enhancement of BCAA catabolism.

Semi-Rational Design of Proteus mirabilis l-Amino Acid Deaminase for Expanding Its Substrate Specificity in α-Keto Acid Synthesis from l-Amino Acids

l-amino acid deaminases (LAADs) are flavoenzymes that catalyze the stereospecific oxidative deamination of l-amino acids into α-keto acids, which are widely used in the pharmaceutical, food, chemical, and cosmetic industries. However, the substrate specificity of available LAADs is limited, and most substrates are concentrated on several bulky or basic l-amino acids. In this study, we employed a LAAD from Proteus mirabilis (PmiLAAD) and broadened its substrate specificity using a semi-rational design strategy. Molecular docking and alanine scanning identified F96, Q278, and E417 as key residues around the substrate-binding pocket of PmiLAAD. Site-directed saturation mutagenesis identified E417 as the key site for substrate specificity expansion. Expansion of the substrate channel with mutations of E417 (E417L, E417A) improved activity toward the bulky substrate l-Trp, and mutation of E417 to basic amino acids (E417K, E417H, E417R) enhanced the universal activity toward various l-amino acid substrates. The variant PmiLAADE417K showed remarkable catalytic activity improvement on seven substrates (l-Ala, l-Asp, l-Ile, l-Leu, l-Phe, l-Trp, and l-Val). The catalytic efficiency improvement obtained by E417 mutation may be attributed to the expansion of the entrance channel and its electrostatic interactions. These PmiLAAD variants with a broadened substrate spectrum can extend the application potential of LAADs.