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 Molecular insights into the inhibition of glutamate dehydrogenase (GDH) by the dicarboxylic acid metabolites

2021 ◽  
Author(s):  
Barsa Kanchan Jyotshna Godsora ◽  
Prem Prakash ◽  
Narayan S. Punekar ◽  
Prasenjit Bhaumik
Keyword(s):  
Dicarboxylic Acid ◽  
Glutamate Dehydrogenase ◽  
Active Site ◽  
Binding Pocket ◽  
Metabolic Enzyme ◽  
Carbon And Nitrogen ◽  
Carboxylate Groups ◽  
Lysine Residues ◽  
Acid Metabolites ◽  
Living Organisms

Glutamate dehydrogenase (GDH) is a salient metabolic enzyme which catalyzes the NAD+- or NADP+-dependent reversible conversion of α-ketoglutarate (AKG) to L-glutamate; and thereby connects the carbon and nitrogen metabolism cycles in all living organisms. The function of GDH is extensively regulated by both metabolites (citrate, succinate, etc.) and non-metabolites (ATP, NADH, etc.) but sufficient molecular evidences are lacking to rationalize the inhibitory effects by the metabolites. We have expressed and purified NADP+-dependent Aspergillus terreus GDH (AtGDH) in recombinant form. Succinate, malonate, maleate, fumarate and tartrate independently inhibit the activity of AtGDH to different extents. The crystal structures of AtGDH complexed with the dicarboxylic acid metabolites and the coenzyme NADPH have been determined. Although AtGDH structures are not complexed with substrate; surprisingly, they acquire super closed conformation like previously reported for substrate and coenzyme bound catalytically competent Aspergillus niger GDH (AnGDH). These dicarboxylic acid metabolites partially occupy the same binding pocket as substrate; but interact with varying polar interactions and the coenzyme NADPH binds to the Domain-II of AtGDH. The low inhibition potential of tartrate as compared to other dicarboxylic acid metabolites is due to its weaker interactions of carboxylate groups with AtGDH. Our results suggest that the length of carbon skeleton and positioning of the carboxylate groups of inhibitors between two conserved lysine residues at the GDH active site might be the determinants of their inhibitory potency. Molecular details on the dicarboxylic acid metabolites bound AtGDH active site architecture presented here would be applicable to GDHs in general.

scholarly journals A Legionella effector ADP-ribosyltransferase inactivates glutamate dehydrogenase

2021 ◽  
pp. 100301
Author(s):  
Miles H. Black ◽  
Adam Osinski ◽  
Gina J. Park ◽  
Marcin Gradowski ◽  
Kelly A. Servage ◽  
...  
Keyword(s):  
Glutamate Dehydrogenase ◽  
Adp Ribosyltransferase

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 Molecular structural diversity of mitochondrial cardiolipins

2018 ◽  
Vol 115 (16) ◽  
pp. 4158-4163 ◽  
Author(s):  
Gregor Oemer ◽  
Katharina Lackner ◽  
Katharina Muigg ◽  
Gerhard Krumschnabel ◽  
Katrin Watschinger ◽  
...  
Keyword(s):  
Molecular Species ◽  
High Performance ◽  
Biological Diversity ◽  
Structural Diversity ◽  
Fatty Acyl ◽  
Model Organisms ◽  
Specific Class ◽  
Accurate Identification ◽  
Individual Molecular Species ◽  
Mass Spectral

Current strategies used to quantitatively describe the biological diversity of lipids by mass spectrometry are often limited in assessing the exact structural variability of individual molecular species in detail. A major challenge is represented by the extensive isobaric overlap present among lipids, hampering their accurate identification. This is especially true for cardiolipins, a mitochondria-specific class of phospholipids, which are functionally involved in many cellular functions, including energy metabolism, cristae structure, and apoptosis. Substituted with four fatty acyl side chains, cardiolipins offer a particularly high potential to achieve complex mixtures of molecular species. Here, we demonstrate how systematically generated high-performance liquid chromatography-mass spectral data can be utilized in a mathematical structural modeling approach, to comprehensively analyze and characterize the molecular diversity of mitochondrial cardiolipin compositions in cell culture and disease models, cardiolipin modulation experiments, and a broad variety of frequently studied model organisms.

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.

scholarly journals The gdhB Gene of Pseudomonas aeruginosaEncodes an Arginine-Inducible NAD+-Dependent Glutamate Dehydrogenase Which Is Subject to Allosteric Regulation

2001 ◽  
Vol 183 (2) ◽  
pp. 490-499 ◽  
Author(s):  
Chung-Dar Lu ◽  
Ahmed T. Abdelal
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Glutamate Dehydrogenase ◽  
Legionella Pneumophila ◽  
Caulobacter Crescentus ◽  
Sodium Dodecyl ◽  
Sequence Information ◽  
Saturation Curve ◽  
Moderate Degree ◽  
Amino Terminal

ABSTRACT The NAD+-dependent glutamate dehydrogenase (NAD-GDH) from Pseudomonas aeruginosa PAO1 was purified, and its amino-terminal amino acid sequence was determined. This sequence information was used in identifying and cloning the encodinggdhB gene and its flanking regions. The molecular mass predicted from the derived sequence for the encoded NAD-GDH was 182.6 kDa, in close agreement with that determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme (180 kDa). Cross-linking studies established that the native NAD-GDH is a tetramer of equal subunits. Comparison of the derived amino acid sequence of NAD-GDH from P. aeruginosa with the GenBank database showed the highest homology with hypothetical polypeptides from Pseudomonas putida, Mycobacterium tuberculosis, Rickettsia prowazakii, Legionella pneumophila, Vibrio cholerae, Shewanella putrefaciens, Sinorhizobium meliloti, andCaulobacter crescentus. A moderate degree of homology, primarily in the central domain, was observed with the smaller tetrameric NAD-GDH (protomeric mass of 110 kDa) fromSaccharomyces cerevisiae or Neurospora crassa. Comparison with the yet smaller hexameric GDH (protomeric mass of 48 to 55 kDa) of other prokaryotes yielded a low degree of homology that was limited to residues important for binding of substrates and for catalytic function. NAD-GDH was induced 27-fold by exogenous arginine and only 3-fold by exogenous glutamate. Primer extension experiments established that transcription of gdhB is initiated from an arginine-inducible promoter and that this induction is dependent on the arginine regulatory protein, ArgR, a member of the AraC/XyIS family of regulatory proteins. NAD-GDH was purified to homogeneity from a recombinant strain of P. aeruginosa and characterized. The glutamate saturation curve was sigmoid, indicating positive cooperativity in the binding of glutamate. NAD-GDH activity was subject to allosteric control by arginine and citrate, which function as positive and negative effectors, respectively. Both effectors act by influencing the affinity of the enzyme for glutamate. NAD-GDH from this organism differs from previously characterized enzymes with respect to structure, protomer mass, and allosteric properties indicate that this enzyme represents a novel class of microbial glutamate dehydrogenases.

scholarly journals Molecular Dynamics Simulation Framework to Probe the Binding Hypothesis of CYP3A4 Inhibitors

2019 ◽  
Vol 20 (18) ◽  
pp. 4468 ◽  
Author(s):  
Kiani ◽  
Ranaghan ◽  
Jabeen ◽  
Mulholland
Keyword(s):  
Molecular Dynamics ◽  
Free Energy ◽  
Binding Free Energy ◽  
Binding Pocket ◽  
Binding Energies ◽  
Dynamics Simulation ◽  
Metabolic Enzyme ◽  
New Chemical Entities ◽  
Cyp3a4 Inhibitors ◽  
Cytochrome P450 Family

The Cytochrome P450 family of heme-containing proteins plays a major role in catalyzing phase I metabolic reactions, and the CYP3A4 subtype is responsible for the metabolism of many currently marketed drugs. Additionally, CYP3A4 has an inherent affinity for a broad spectrum of structurally diverse chemical entities, often leading to drug–drug interactions mediated by the inhibition or induction of the metabolic enzyme. The current study explores the binding of selected highly efficient CYP3A4 inhibitors by docking and molecular dynamics (MD) simulation protocols and their binding free energy calculated using the WaterSwap method. The results indicate the importance of binding pocket residues including Phe57, Arg105, Arg106, Ser119, Arg212, Phe213, Thr309, Ser312, Ala370, Arg372, Glu374, Gly481 and Leu483 for interaction with CYP3A4 inhibitors. The residue-wise decomposition of the binding free energy from the WaterSwap method revealed the importance of binding site residues Arg106 and Arg372 in the stabilization of all the selected CYP3A4-inhibitor complexes. The WaterSwap binding energies were further complemented with the MM(GB/PB)SA results and it was observed that the binding energies calculated by both methods do not differ significantly. Overall, our results could guide towards the use of multiple computational approaches to achieve a better understanding of CYP3A4 inhibition, subsequently leading to the design of highly specific and efficient new chemical entities with suitable ADMETox properties and reduced side effects.

Sign in / Sign up

Export Citation Format

Share Document