Leucine dehydrogenase (LDH) is the key rate-limiting enzyme in the production of L-2-aminobutyric acid (L-2-ABA). In this study, we modified the C-terminal Loop region of this enzyme to improve the specific enzyme activity and stability for efficient synthesis of L-2-ABA. Using molecular dynamics simulation of LDH, we analyzed the change of root mean square fluctuation (RMSF), rationally designed the Loop region with greatly fluctuated RMSF, and obtained a mutant EsLDHD2 with a specific enzyme activity 23.2% higher than that of the wild type. Since the rate of the threonine deaminase-catalyzed reaction converting L-threonine into 2-ketobutyrate was so fast, the multi-enzyme cascade catalysis system became unbalanced. Therefore, the LDH and the formate dehydrogenase were double copied in a new construct E. coli BL21/pACYCDuet-RM. Compared with E. coli BL21/pACYCDuet-RO, the molar conversion rate of L-2-ABA increased by 74.6%. The whole cell biotransformation conditions were optimized and the optimal pH, temperature and substrate concentration were 7.5, 35 °C and 80 g/L, respectively. Under these conditions, the molar conversion rate was higher than 99%. Finally, 80 g and 40 g L-threonine were consecutively fed into a 1 L reaction mixture under the optimal conversion conditions, producing 97.9 g L-2-ABA. Thus, this strategy provides a green and efficient synthesis of L-2-ABA, and has great industrial application potential.
Aminobutyrates ; Escherichia coli/genetics* ; Leucine Dehydrogenase/genetics* ; Threonine Dehydratase

In this study, Escherichia coli BL21 (DE3) was used as the host to construct 2 recombinant E. coli strains that co-expressed leucine dehydrogenase (LDH, Bacillus cereus)/formate dehydrogenase (FDH, Ancylobacter aquaticus), or leucine dehydrogenase (LDH, Bacillus cereus)/alcohol dehydrogenase (ADH, Rhodococcus), respectively. L-2-aminobutyric acid was then synthesized by L-threonine deaminase (L-TD) with LDH-FDH or LDH-ADH by coupling with two different NADH regeneration systems. LDH-FDH process and LDH-ADH process were optimized and compared with each other. The optimum reaction pH of LDH-FDH process was 7.5, and the optimum reaction temperature was 35 °C. After 28 h, the concentration of L-2-aminobutyric acid was 161.8 g/L with a yield of 97%, when adding L-threonine in batches for controlling 2-ketobutyric acid concentration less than 15 g/L and using 50 g/L ammonium formate, 0.3 g/L NAD+, 10% LDH-FDH crude enzyme solution (V/V) and 7 500 U/L L-TD. The optimum reaction pH of LDH-ADH process was 8.0, and the optimum reaction temperature was 35 °C. After 24 h, the concentration of L-2-aminobutyric acid was 119.6 g/L with a yield of 98%, when adding L-threonine and isopropanol (1.2 times of L-threonine) in batches for controlling 2-ketobutyric acid concentration less than 15 g/L, removing acetone in time and using 0.3 g/L NAD⁺, 10% LDH-ADH crude enzyme solution (V/V) and 7 500 U/L L-TD. The process and results used in this paper provide a reference for the industrialization of L-2-aminobutyric acid.
Aminobutyrates ; metabolism ; Escherichia coli ; genetics ; Formate Dehydrogenases ; metabolism ; Leucine Dehydrogenase ; metabolism ; NAD ; metabolism

The hyperinsulinism/hyperammonemia (HI/HA) syndrome is a form of congenital hyperinsulinism. The children with HI/HA syndrome present recurrent symptomatic hypoglycemia and asymptomatic, persistent hyperammonemia, caused by mutations of the GLUD1 encoding the mitochondrial enzyme, glutamate dehydrogenase (GDH). The mutations impair sensitivity to the inhibition of GTP (guanosine triphosphate), which results in stimulation of insulin secretion from pancreatic beta-cells and increased rates of ammonia production. Leucine is known to mediate the insulin secretion. We report HI/HA syndrome with a 12-month-old male who had intermittent hypoglycemia. We revealed characteristic clinical findings of hypoglycemia induced by oral administration of protein in this patient who had mutations of GLUD1 (S445L).
Administration, Oral ; Ammonia ; Child ; Congenital Hyperinsulinism ; Glutamate Dehydrogenase ; Guanosine Triphosphate ; Humans ; Hyperammonemia ; Hyperinsulinism ; Hypoglycemia ; Infant ; Insulin ; Leucine ; Male

Maple syrup urine disease (MSUD) is an autosomal recessive disorder. Impaired activity of the branched-chain 2-oxo acid dehydrogenase complex causes accumulation of branched-chain L-amino and 2-oxo acid that can exert neurotoxic effects. MSUD presents heterogeneous clinical and molecular phenotypes. Severity of the disease, ranging from classical to mild variant types, is commonly classified on the basis of indirect parameters, e.g. onset, leucine tolerance and/or residual enzyme activity in cell. Since early 1990's, tandem mass spectrometry has been applied to newborn screening, because it is amenable to population-wide testing for a large number of disorders of fatty acid, organic acid, and amino acid metabolism. And so, we report a case of MSUD in 15 days old boy detected by newborn screening using tandem mass spectrometry.
3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide) ; Acer* ; Humans ; Infant, Newborn* ; Leucine ; Male ; Maple Syrup Urine Disease* ; Mass Screening* ; Metabolism ; Phenotype ; Tandem Mass Spectrometry*

L-2-aminobutyric acid (L-ABA) is an important chemical raw material and chiral pharmaceutical intermediate. The aim of this study was to develop an efficient method for L-ABA production from L-threonine using a trienzyme cascade route with Threonine deaminase (TD) from Escherichia. coli, Leucine dehydrogenase (LDH) from Bacillus thuringiensis and Formate dehydrogenase (FDH) from Candida boidinii. In order to simplify the production process, the activity ratio of TD, LDH and FDH was 1:1:0.2 after combining different activity ratios in the system in vitro. The above ratio was achieved in the recombinant strain E. coli 3FT+L. Moreover, the transformation conditions were optimized. Finally, we achieved L-ABA production of 68.5 g/L with a conversion rate of 99.0% for 12 h in a 30-L bioreactor by whole-cell catalyst. The environmentally safe and efficient process route represents a promising strategy for large-scale L-ABA production in the future.
Aminobutyrates ; chemical synthesis ; Bacillus thuringiensis ; enzymology ; Candida ; enzymology ; Escherichia coli ; enzymology ; Formate Dehydrogenases ; metabolism ; Leucine Dehydrogenase ; metabolism ; Threonine ; metabolism ; Threonine Dehydratase ; metabolism

BACKGROUND/OBJECTIVES: The study was conducted to evaluate the effects of dietary leucine supplementation on mitochondrial biogenesis and energy metabolism in the liver of normal birth weight (NBW) and intrauterine growth-retarded (IUGR) weanling piglets. MATERIALS/METHODS: A total of sixteen pairs of NBW and IUGR piglets from sixteen sows were selected according to their birth weight. At postnatal day 14, all piglets were weaned and fed either a control diet or a leucine-supplemented diet for 21 d. Thereafter, a 2 × 2 factorial experimental design was used. Each treatment consisted of eight replications with one piglet per replication. RESULTS: Compared with NBW piglets, IUGR piglets had a decreased (P < 0.05) hepatic adenosine triphosphate (ATP) content. Also, IUGR piglets exhibited reductions (P < 0.05) in the activities of hepatic mitochondrial pyruvate dehydrogenase (PDH), citrate synthase (CS), α-ketoglutarate dehydrogenase (α-KGDH), malate dehydrogenase (MDH), and complexes I and V, along with decreases (P < 0.05) in the concentration of mitochondrial DNA (mtDNA) and the protein expression of hepatic peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α). Dietary leucine supplementation increased (P < 0.05) the content of ATP, and the activities of CS, α-KGDH, MDH, and complex V in the liver of piglets. Furthermore, compared to those fed a control diet, piglets given a leucine-supplemented diet exhibited increases (P < 0.05) in the mtDNA content and in the mRNA expressions of sirtuin 1, PGC-1α, nuclear respiratory factor 1, mitochondrial transcription factor A, and ATP synthase, H+ transporting, mitochondrial F1 complex, β polypeptide in liver. CONCLUSIONS: Dietary leucine supplementation may exert beneficial effects on mitochondrial biogenesis and energy metabolism in NBW and IUGR weanling piglets.
Adenosine Triphosphate ; Birth Weight* ; Citrate (si)-Synthase ; Diet ; DNA, Mitochondrial ; Energy Metabolism* ; Fetal Growth Retardation ; Leucine* ; Liver ; Malate Dehydrogenase ; Nuclear Respiratory Factor 1 ; Organelle Biogenesis* ; Oxidoreductases ; Parturition* ; Peroxisomes ; Pyruvic Acid ; Research Design ; RNA, Messenger ; Sirtuin 1 ; Transcription Factors

PURPOSE: Oxygen is indispensable for survival and aerobic metabolism in all mammalian cells. Inadequate oxygen triggers a multifaceted cellular response negatively impacting important physiological functions which are observed in clinical diseases such as stroke, drowning, cardiac arrest, hazardous gas poisoning, myocardial infarction and vascular dementia. In this study, we investigated the neuroprotective effect of a synthetic delta-opioid agonist, [D-Ala2, D-Leu5] enkephalin (DADLE), and its role in ischemic neuronal injury. METHODS: This experiment was conducted in vitro using a primary culture of rat cortical neurons. Ischemia induction was performed using a hypoxic chamber. To test the degree of neuronal viability, as protected by delta-opioid stimulation with DADLE under ischemia, we used three independent approaches including a lactate dehydrogenase assay, MTT assay, and an immunofluorescent staining assay for viable cells. In addition, the gene expressions of caspase-3 and heat shock protein 70 were analyzed using real-time PCR. RESULTS: Incubation of the cortical neurons with DADLE protected them from ischemia-induced cytotoxicity, as observed by all three independent viability assays. Also, we found that its neuroprotective effect might be related with suppression of the caspase-3 gene. CONCLUSION: The results of this study suggested that DADLE exhibits a neuroprotective effect against ischemia-induced neuronal cell death.
Animals ; Caspase 3 ; Cell Death ; Dementia, Vascular ; Drowning ; Enkephalin, Leucine-2-Alanine ; Enkephalins ; Gas Poisoning ; Gene Expression ; Heart Arrest ; HSP70 Heat-Shock Proteins ; Ischemia ; L-Lactate Dehydrogenase ; Myocardial Infarction ; Neurons ; Neuroprotective Agents ; Oxygen ; Rats ; Stroke

PURPOSE: Oxygen is indispensable for survival and aerobic metabolism in all mammalian cells. Inadequate oxygen triggers a multifaceted cellular response negatively impacting important physiological functions which are observed in clinical diseases such as stroke, drowning, cardiac arrest, hazardous gas poisoning, myocardial infarction and vascular dementia. In this study, we investigated the neuroprotective effect of a synthetic delta-opioid agonist, [D-Ala2, D-Leu5] enkephalin (DADLE), and its role in ischemic neuronal injury. METHODS: This experiment was conducted in vitro using a primary culture of rat cortical neurons. Ischemia induction was performed using a hypoxic chamber. To test the degree of neuronal viability, as protected by delta-opioid stimulation with DADLE under ischemia, we used three independent approaches including a lactate dehydrogenase assay, MTT assay, and an immunofluorescent staining assay for viable cells. In addition, the gene expressions of caspase-3 and heat shock protein 70 were analyzed using real-time PCR. RESULTS: Incubation of the cortical neurons with DADLE protected them from ischemia-induced cytotoxicity, as observed by all three independent viability assays. Also, we found that its neuroprotective effect might be related with suppression of the caspase-3 gene. CONCLUSION: The results of this study suggested that DADLE exhibits a neuroprotective effect against ischemia-induced neuronal cell death.
Animals ; Caspase 3 ; Cell Death ; Dementia, Vascular ; Drowning ; Enkephalin, Leucine-2-Alanine ; Enkephalins ; Gas Poisoning ; Gene Expression ; Heart Arrest ; HSP70 Heat-Shock Proteins ; Ischemia ; L-Lactate Dehydrogenase ; Myocardial Infarction ; Neurons ; Neuroprotective Agents ; Oxygen ; Rats ; Stroke

Maple syrup urine disease (MSUD) is a disorder that involves the metabolism of branched chain amino acids, arising from a defect in branched-chain alpha-keto acid dehydrogenase complex. Mutations have been identified in the BCKDHA, BCKDHB, or DBT genes, which encode different subunits of the BCKDH complex. Although encephalopathy and progressive neurodegeneration are its major manifestations, the severity of the disease may range from the severe classic type to milder intermediate variants. We report two Korean siblings with the milder intermediate MSUD who were diagnosed with MSUD by a combination of newborn screening tests using tandem mass spectrometry and family genetic screening for MSUD. At diagnosis, the patients' plasma levels were elevated for leucine, isoleucine, valine, and alloisoleucine, and branched-chain alpha-keto acids and branched-chain alpha-hydroxy acids were detected in their urine. BCKDHA, BCKDHB, and DBT analysis was performed, and two novel mutations were identified in BCKDHB. Our patients were thought to have the milder intermediate variant of MSUD, rather than the classic form. Although MSUD is a typical metabolic disease with poor prognosis, better outcomes can be expected if early diagnosis and prompt management are provided, particularly for milder forms of the disease.
3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide) ; Amino Acids ; Diagnosis ; Early Diagnosis ; Genetic Testing ; Humans ; Infant, Newborn ; Isoleucine ; Leucine ; Maple Syrup Urine Disease* ; Mass Screening ; Metabolic Diseases ; Metabolism ; Plasma ; Prognosis ; Siblings* ; Tandem Mass Spectrometry ; Valine

The principal objective of this study was to determine the effects of leucine on body weight reduction in high fat diet-induced overweight rats. To induce overweight, six-month-old male Sprague-Dawley rats (n = 80) were divided into 8 groups; one group of 10 rats was fed on a normal fat diet and the remaining 70 rats were fed on a high-fat diet (40% of energy as fat) for 14 weeks. Then, 10 rats fed on the normal fat diet and another 10 rats fed on the high fat diet were sacrificed to identify overweight induction. The remaining 60 rats were divided randomly into 6 groups according to body weight and fed on one of the diets with different dietary fat levels (9.6% or 40% of energy as fat) and leucine levels (0, 0.6 or 1.2 g/kg BW) for the following 5 weeks of experiments. The body weight loss in the Leu-administered groups (0.6 g, 1.2 g/kg BW) was significantly higher than those of Leu non-administered groups. The perirenal fat pad weights in the Leu-administered groups were significantly lower than those of the Leu non-administered groups. Of the hepatic enzymes, glucose-6-phosphate dehydrogenase (G6PDH) activities were reduced significantly in the Leu-administered groups than in the Leu non-administered groups. With the oral glucose tolerance test (OGTT), the incremental areas under the curve of the glucose response (IAUC) of the Leu-administered groups were significantly lower than those of the Leu non-administered groups. The fasting glucose concentration and HOMA-IR of the Leu-administered groups were significantly lower than those of the Leu non-administered groups. In conclusion, the results of this study suggest that one of the possible mechanisms of leucine in the observed body weight reduction might involve the inhibition of lipogenic enzyme activities such as glucose-6-phosphate dehydrogenase, rather than the activation of lipolysis enzymes. Additionally, leucine adminstration resulted in improved glucose metabolism.
Adipose Tissue ; Animals ; Blood Glucose ; Body Weight ; Diet ; Diet, High-Fat ; Dietary Fats ; Fasting ; Glucose ; Glucose Tolerance Test ; Glucosephosphate Dehydrogenase ; Humans ; Leucine ; Lipolysis ; Male ; Overweight ; Rats ; Rats, Sprague-Dawley ; Weights and Measures