Properties of NAD-dependent glutamate dehydrogenase from the tylosin producer Streptomyces fradiae

1997 ◽  
Vol 43 (11) ◽  
pp. 1005-1010 ◽  
Author(s):  
Kien Trung Nguyen ◽  
Lieu Thi Nguyen ◽  
Jan Kopecký ◽  
Vladislav Běhal
Keyword(s):  
Key Words ◽  
Glutamate Dehydrogenase ◽  
Reductive Amination ◽  
Divalent Cations ◽  
Ammonium Assimilation ◽  
Alkaline Ph ◽  
Streptomyces Fradiae ◽  
Oxidative Deamination

Glutamate dehydrogenase is an enzyme responsible for ammonium assimilation and glutamate catabolism in organisms. The tylosin producer Streptomyces fradiae possesses both NADP- and NAD-dependent glutamate dehydrogenases. The latter enzyme was purified 498-fold with a 7.5% recovery by a six-step protocol. The enzyme is composed of two subunits, each of Mr 47 000, and could form active aggregates of four or eight subunits. Its activity was inactivated by alkaline pH or temperatures of −20 °C or above 40 °C. Activities assayed in the direction of oxidative deamination and reductive amination were optimal at pH 9.2 and 8.8, respectively, and at temperatures of 30–35 °C. No activity was found when NAD(H) was replaced with NADP(H). The Km values were 32.2 mM for L-glutamate, 0.3 mM for NAD+, 3.4 mM for 2-ketoglutarate, 14.2 mM for NH4+, and 0.05 mM for NADH. Deamination activity was partially inhibited by adenyl nucleotides and several divalent cations; amination activity was not affected by the nucleotides but significantly inhibited by Cu2+ or Ni2+.Key words: Streptomyces fradiae, NAD-dependent glutamate dehydrogenase, purification, properties.

scholarly journals Anticonvulsant Drugs, Brain Glutamate Dehydrogenase Activity and Oxygen Consumption

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Lourdes A. Vega Rasgado ◽  
Guillermo Ceballos Reyes ◽  
Fernando Vega-Díaz
Keyword(s):  
Oxygen Consumption ◽  
Glutamate Dehydrogenase ◽  
Reductive Amination ◽  
Pyridoxal Phosphate ◽  
Neuronal Excitability ◽  
Aminooxyacetic Acid ◽  
Oxidative Deamination ◽  
Key Enzyme

Glutamate dehydrogenase (GDH, E.C. 1.4.1.3.) is a key enzyme for the biosynthesis and modulation of glutamate (GLU) metabolism and an indirect γ-aminobutyric acid (GABA) source, here we studied the effect of anticonvulsants such as pyridoxal phosphate (PPAL), aminooxyacetic acid (AAOA), and hydroxylamine (OHAMINE) on GDH activity in mouse brain. Moreover, since GLU is a glucogenic molecule and anoxia is a primary cause of convulsions, we explore the effect of these drugs on oxygen consumption. Experiments were performed in vitro as well as in vivo for both oxidative deamination of GLU and reductive amination of α-ketoglutarate (αK). Results in vitro showed that PPAL decreased oxidative deamination of GLU and oxygen consumption, whereas AAOA and OHAMINE inhibited GDH activity competitively and also inhibited oxygen consumption when αK reductive amination was carried out. In contrast, results showed that in vivo, all anticonvulsants enhanced GLU utilization by GDH and also decreased oxygen consumption. Together, results suggest that GDH activity has repercussions on oxygen consumption, which may indicate that the enzyme activity is highly regulated by energy requirements for metabolic activity. Besides, GDH may participate in regulation of GLU and, indirectly GABA levels, hence in neuronal excitability, becoming a key enzyme in seizures mechanism.

scholarly journals Modulation of brain glutamate dehydrogenase as a tool for controlling seizures

2015 ◽  
Vol 65 (4) ◽  
pp. 443-452 ◽  
Author(s):  
Lourdes A. Vega Rasgado ◽  
Guillermo Ceballos Reyes ◽  
Fernando Vega Díaz
Keyword(s):  
Oxygen Consumption ◽  
Glutamate Dehydrogenase ◽  
Reductive Amination ◽  
Neuronal Excitability ◽  
Ex Vivo ◽  
Oxidative Deamination ◽  
Excitatory Neurotransmitter ◽  
Mice Brain

Abstract Glutamate (Glu) is a major excitatory neurotransmitter involved in epilepsy. Glu is synthesized by glutamate dehydrogenase (GDH, E.C. 1.4.1.3) and dysfunction of the enzymatic activity of GDH is associated with brain pathologies. The main goal of this work is to establish the role of GDH in the effects of antiepileptic drugs (AEDs) such as valproate (VALP), diazepam (DIAZ) and diphenylhydantoin (DPH) and its repercussions on oxygen consumption. Oxidative deamination of Glu and reductive amination of aketoglutarate (αK) in mice brain were investigated. Our results show that AEDs decrease GDH activity and oxygen consumption in vitro. In ex vivo experiments, AEDs increased GDH activity but decreased oxygen consumption during Glu oxidative deamination. VALP and DPH reversed the increase in reductive amination of αK caused by the chemoconvulsant pentylenetetrazol. These results suggest that AEDs act by modulating brain GDH activity, which in turn decreased oxygen consumption. GDH represents an important regulation point of neuronal excitability, and modulation of its activity represents a potential target for metabolic treatment of epilepsy and for the development of new AEDs.

scholarly journals Hetero-oligomeric glutamate dehydrogenase from Thermus thermophilus

Microbiology ◽  
2010 ◽  
Vol 156 (12) ◽  
pp. 3801-3813 ◽  
Author(s):  
Takeo Tomita ◽  
Takashi Miyazaki ◽  
Junichi Miyazaki ◽  
Tomohisa Kuzuyama ◽  
Makoto Nishiyama
Keyword(s):  
Amino Acids ◽  
Glutamate Dehydrogenase ◽  
Reductive Amination ◽  
Thermus Thermophilus ◽  
Thermophilic Bacterium ◽  
Oxidative Deamination ◽  
Turnover Number ◽  
Catalytic Subunits ◽  
His Tag ◽  
Hydrophobic Amino Acids

An extremely thermophilic bacterium, Thermus thermophilus, possesses two glutamate dehydrogenase (GDH) genes, gdhA and gdhB, putatively forming an operon on the genome. To elucidate the functions of these genes, the gene products were purified and characterized. GdhA showed no GDH activity, while GdhB showed GDH activity for reductive amination 1.3-fold higher than that for oxidative deamination. When GdhA was co-expressed with His-tag-fused GdhB, GdhA was co-purified with His-tagged GdhB. Compared with GdhB alone, co-purified GdhA–GdhB had decreased reductive amination activity and increased oxidative deamination activity, resulting in a 3.1-fold preference for oxidative deamination over reductive amination. Addition of hydrophobic amino acids affected the GDH activity of the co-purified GdhA–GdhB hetero-complex. Among the amino acids, leucine had the largest effect on activity: addition of 1 mM leucine elevated the GDH activity of the co-purified GdhA–GdhB by 974 and 245 % for reductive amination and oxidative deamination, respectively, while GdhB alone did not show such marked activation by leucine. Kinetic analysis revealed that the elevation of GDH activity by leucine is attributable to the enhanced turnover number of GDH. In this hetero-oligomeric GDH system, GdhA and GdhB act as regulatory and catalytic subunits, respectively, and GdhA can modulate the activity of GdhB through hetero-complex formation, depending on the availability of hydrophobic amino acids. This study provides the first finding, to our knowledge, of a hetero-oligomeric GDH that can be regulated allosterically.

A Legionella effector ADP-ribosyltransferase inactivates glutamate dehydrogenase

2020 ◽  
Author(s):  
Miles H. Black ◽  
Adam Osinski ◽  
Marcin Gradowski ◽  
Kelly A. Servage ◽  
Krzysztof Pawłowski ◽  
...  
Keyword(s):  
Glutamate Dehydrogenase ◽  
Legionella Pneumophila ◽  
Structural Diversity ◽  
Binding Pocket ◽  
Metabolic Enzyme ◽  
Specific Class ◽  
Oxidative Deamination ◽  
Natural Hosts ◽  
Adp Ribosyltransferase ◽  
Bioinformatic Approach

AbstractADP-ribosyltransferases (ARTs) are a widespread superfamily of enzymes frequently employed in pathogenic strategies of bacteria. Legionella pneumophila, the causative agent of Legionnaire’s disease, has acquired over 330 translocated effectors that showcase remarkable biochemical and structural diversity. Here we took a bioinformatic approach to search the Legionella effector repertoire for additional divergent members of the ART superfamily and identified an ART domain in Lpg0181. We show that L. pneumophila Lpg0181 targets a specific class of 120-kDa NAD+-dependent glutamate dehydrogenase (GDH) enzymes found in fungi and protists, including many natural hosts of Legionella. Lpg0181 targets a conserved arginine residue in the NAD+ -binding pocket of GDH, thereby blocking oxidative deamination of glutamate. While intracellular pathogens employ diverse virulence mechanisms to overcome host-limited nutrient availability, Lpg0181 is––to the best of our knowledge––the first example of a Legionella effector which directly targets a host metabolic enzyme.

scholarly journals Effect of Water Chemistry on Antimony Removal by Chemical Coagulation: Implications of ζ-Potential and Size of Precipitates

2019 ◽  
Vol 20 (12) ◽  
pp. 2945 ◽  
Author(s):  
Muhammad Ali Inam ◽  
Rizwan Khan ◽  
Muhammad Akram ◽  
Sarfaraz Khan ◽  
Ick Tae Yeom
Keyword(s):  
Water Chemistry ◽  
Divalent Cations ◽  
Chemical Properties ◽  
Point Of Zero Charge ◽  
Hydrodynamic Diameter ◽  
Alkaline Ph ◽  
Solution Ph ◽  
Ζ Potential ◽  
Wide Group ◽  
Co Precipitation

The process of coagulation and precipitation affect the fate and mobility of antimony (Sb) species in drinking water. Moreover, the solubility and physico-chemical properties of the precipitates may be affected by the media chemistry. Accordingly, the present study aimed to investigate the removal of Sb(III, V) species by ferric chloride coagulation under various water chemistry influences with a particular focus on the role of the properties of the precipitates. The results indicated that the amount of Sb(III) removed increased with increasing solution pH, showing the insignificant effects of the hydrodynamic diameter (HDD) and ζ-potential of the precipitates. However, no Sb(V) removal occurred at alkaline pH values, while a highly negative ζ-potential and the complete dissolution of precipitates were observed in the aqueous solution. The solution pH was also useful in determining the dominant coagulation mechanisms, such as co-precipitation and adsorption. The Fe solubility substantially affects the Sb removal at a certain pH range, while the HDD of the precipitates plays an insignificant role in Sb removal. The presence of divalent cations brings the ζ-potential of the precipitates close to point of zero charge (pzc), thus enhancing the Sb(V) removal at alkaline pH conditions. Pronounced adverse effects of humic acid were observed on Sb removal, ζ-potential and HDD of the precipitates. In general, this study may provide critical information to a wide group of researchers dealing with environmental protection from heavy metal pollution.

Studies on possible routes of ammonium assimilation in soybean root nodule bacteroids

1973 ◽  
Vol 19 (12) ◽  
pp. 1493-1499 ◽  
Author(s):  
Stanley D. Dunn ◽  
Robert V. Klucas
Keyword(s):  
Glutamine Synthetase ◽  
Nitrogen Assimilation ◽  
Reductive Amination ◽  
Root Nodule ◽  
Root Nodules ◽  
Ammonium Assimilation ◽  
Kinetic Properties ◽  
Transferase Activity ◽  
Soybean Root

Glutamine amide–2-oxoglutarate aminotransferase NAD+ oxidoreductase (GOGAT), glutamine synthetase (GS), glutamate dehydrogenase (GD), and alanine dehydrogenase (AD) were studied in soybean root nodules. GS, GOGAT, and AD were present in bacteroids at levels that could account for ammonium assimilation, but GD activity was quite low. The total activities of GS and GD were higher in the cytosol than in the bacteroids by factors of 20 and 7, respectively, whereas GOGAT was not detected in the cytosol. GS (transferase activity) was inhibited by alanine, CTP, glycine, and tryptophan at 5 mM but was relatively unaffected by asparagine, aspartic acid, CMP, glucosamine, and histidine at 5 mM. GOGAT activity was unaffected by ATP, ADP, 8-hydroxyquinoline, and 1,10-phenanthroline but was inhibited by EDTA, citrate, and parachloromercuribenzoate. GOGAT activity (reductive amination) was also inhibited 97% by preincubation with 10−4 M azaserine for 30 min but GD activity was inhibited only 13%. The apparent Km values for NH4+ by AD was 7.4 × 10−3 M and by GD was 7.3 × 10−2 M while for glutamine by GOGAT it was 9.3 × 10−5 M. Activities and kinetic properties for these enzymes may suggest potential routes of nitrogen assimilation in vivo.

scholarly journals Crystal structure of a chimaeric bacterial glutamate dehydrogenase

2016 ◽  
Vol 72 (6) ◽  
pp. 462-466 ◽  
Author(s):  
Tânia Oliveira ◽  
Michael A. Sharkey ◽  
Paul C. Engel ◽  
Amir R. Khan
Keyword(s):  
Crystal Structure ◽  
Glutamate Dehydrogenase ◽  
Rigid Bodies ◽  
Nucleotide Binding Domain ◽  
Oxidative Deamination ◽  
E Coli ◽  
Cofactor Binding ◽  
Signature Sequences ◽  
Clostridium Symbiosum ◽  
Domain I

Glutamate dehydrogenases (EC 1.4.1.2–4) catalyse the oxidative deamination of L-glutamate to α-ketoglutarate using NAD(P)+as a cofactor. The bacterial enzymes are hexameric, arranged with 32 symmetry, and each polypeptide consists of an N-terminal substrate-binding segment (domain I) followed by a C-terminal cofactor-binding segment (domain II). The catalytic reaction takes place in the cleft formed at the junction of the two domains. Distinct signature sequences in the nucleotide-binding domain have been linked to the binding of NAD+versusNADP+, but they are not unambiguous predictors of cofactor preference. In the absence of substrate, the two domains move apart as rigid bodies, as shown by the apo structure of glutamate dehydrogenase fromClostridium symbiosum. Here, the crystal structure of a chimaeric clostridial/Escherichia colienzyme has been determined in the apo state. The enzyme is fully functional and reveals possible determinants of interdomain flexibility at a hinge region following the pivot helix. The enzyme retains the preference for NADP+cofactor from the parentE. colidomain II, although there are subtle differences in catalytic activity.

Sign in / Sign up

Export Citation Format

Share Document