Structural insights into the allosteric site of Arabidopsis NADP-malic enzyme 2: role of the second sphere residues in the regulatory signal transmission

Human mitochondrial NAD(P)+-dependent malate dehydrogenase (decarboxylating) (malic enzyme) can be specifically and allosterically activated by fumarate. X-ray crystal structures have revealed conformational changes in the enzyme in the absence and in the presence of fumarate. Previous studies have indicated that fumarate is bound to the allosteric pocket via Arg67 and Arg91. Mutation of these residues almost abolishes the activating effect of fumarate. However, these amino acid residues are conserved in some enzymes that are not activated by fumarate, suggesting that there may be additional factors controlling the activation mechanism. In the present study, we tried to delineate the detailed molecular mechanism of activation of the enzyme by fumarate. Site-directed mutagenesis was used to replace Asp102, which is one of the charged amino acids in the fumarate binding pocket and is not conserved in other decarboxylating malate dehydrogenases. In order to explore the charge effect of this residue, Asp102 was replaced by alanine, glutamate or lysine. Our experimental data clearly indicate the importance of Asp102 for activation by fumarate. Mutation of Asp102 to Ala or Lys significantly attenuated the activating effect of fumarate on the enzyme. Kinetic parameters indicate that the effect of fumarate was mainly to decrease the Km values for malate, Mg2+ and NAD+, but it did not notably elevate kcat. The apparent substrate Km values were reduced by increasing concentrations of fumarate. Furthermore, the greatest effect of fumarate activation was apparent at low malate, Mg2+ or NAD+ concentrations. The Kact values were reduced with increasing concentrations of malate, Mg2+ and NAD+. The Asp102 mutants, however, are much less sensitive to regulation by fumarate. Mutation of Asp102 leads to the desensitization of the co-operative effect between fumarate and substrates of the enzyme.

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A Comparative Study of 1D Descriptors Supported CoMFA and CoMSIA QSAR Models to Gain Novel Insights into 1,2,4-Triazoles Acting As Antitubercular Agents

Current Computer - Aided Drug Design ◽

10.2174/1573409916666200302115432 ◽

2020 ◽

Vol 16 ◽

Author(s):

Rajdeep Ray ◽

Gautham Shenoy ◽

N V Ganesh Kumar Tummalapalli

Keyword(s):

External Validation ◽

Antitubercular Activity ◽

New Drugs ◽

Rational Approach ◽

Antitubercular Drugs ◽

Comfa Model ◽

Qsar Models ◽

Structural Insights ◽

The Way

: Tuberculosis is one of the leading cause for deaths due to infectious disease worldwide. There is an urgent need for developing new drugs due to the rising incidents of drug resistance. Triazoles have previously been reported to show antitubercular activity. Various computational tools pave the way for a rational approach in understanding the structural importance of these compounds in inhibiting Mycobacterium tuberculosis growth. The aim of this study is to develop and compare two different QSAR models based on a set of previously reported molecules and use the best one for gaining structural insights in to the Triazole molecules. In the current study, two separate models were generated with CoMFA and CoMSIA descriptors respectively based on a dataset of triazole molecules showing antitubercular activity. Several one dimensional (1D) descriptors were added to each of the models and the validation results and the contour data generated from them were compared. The best model was studied to give a detailed understanding of the triazole molecules and their role in the antitubercular activity.The r2, q2, predicted r2 and SEP (Standard error of prediction) for the CoMFA model were 0.866, 0.573, 0.119 and 0.736 respectively and for the CoMSIA model the r2, q2, predicted r2 and SEP were calculated to be 0.998, 0.634, 0.013 and 0.869 respectively. Although both the QSAR models produced acceptable internal and external validation scores but the CoMSIA results were significantly better. The CoMSIA contours also provided a better match than CoMFA with most of the features of the active compound 30b. Hence, the CoMSIA model was chosen and its contours were explored for gaining structural insights on the triazole molecules. The CoMSIA contours helped us to understand the role of several atoms and groups of the triazole molecules in their biological activity. The possibilities for substitution in the triazole compounds that would enhance the activity were also analysed. Thus, this study paves the way for designing new antitubercular drugs in future.

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The role of malic enzyme in the regulation of lipid accumulation in filamentous fungi

Microbiology ◽

10.1099/13500872-145-8-1911 ◽

1999 ◽

Vol 145 (8) ◽

pp. 1911-1917 ◽

Cited By ~ 147

Author(s):

James P. Wynn ◽

Aidil bin Abdul Hamid ◽

Colin Ratledge

Keyword(s):

Filamentous Fungi ◽

Lipid Accumulation ◽

Malic Enzyme

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Role of Pyruvate Carboxylase, Phosphoenolpyruvate Carboxykinase, and Malic Enzyme during Growth and Sporulation of Bacillus subtilis

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)43509-6 ◽

1973 ◽

Vol 248 (17) ◽

pp. 6062-6070

Author(s):

Martin D. Diesterhaft ◽

Ernst Freese

Keyword(s):

Bacillus Subtilis ◽

Malic Enzyme ◽

Pyruvate Carboxylase ◽

Phosphoenolpyruvate Carboxykinase

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The role of malic enzyme as the provider of NADPH in oleaginous microorganisms: a reappraisal and unsolved problems

Biotechnology Letters ◽

10.1007/s10529-014-1532-3 ◽

2014 ◽

Vol 36 (8) ◽

pp. 1557-1568 ◽

Cited By ~ 134

Author(s):

Colin Ratledge

Keyword(s):

Malic Enzyme ◽

Oleaginous Microorganisms

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Glucose stimulates transcription of fatty acid synthase and malic enzyme in avian hepatocytes

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1998.274.3.e493 ◽

1998 ◽

Vol 274 (3) ◽

pp. E493-E501 ◽

Cited By ~ 2

Author(s):

F. Bradley Hillgartner ◽

Tina Charron

Keyword(s):

Fatty Acid ◽

High Fat Diet ◽

Malic Enzyme ◽

Fatty Acid Synthase ◽

Primary Cultures ◽

Pentose Phosphate ◽

Mrna Abundance ◽

Low Fat Diet ◽

Low Carbohydrate

Transcription of fatty acid synthase (FAS) and malic enzyme (ME) in avian liver is low during starvation or feeding a low-carbohydrate, high-fat diet and high during feeding a high-carbohydrate, low-fat diet. The role of glucose in the nutritional control of FAS and ME was investigated by determining the effects of this metabolic fuel on expression of FAS and ME in primary cultures of chick embryo hepatocytes. In the presence of triiodothyronine, glucose (25 mM) stimulated an increase in the activity and mRNA abundance of FAS and ME. These effects required the phosphorylation of glucose to glucose 6-phosphate but not further metabolism downstream of the aldolase step of the glycolytic pathway. Xylitol mimicked the effects of glucose on FAS and ME expression, suggesting that an intermediate of the pentose phosphate pathway may be involved in mediating this response. The effects of glucose on the mRNA abundance of FAS and ME were accompanied by similar changes in transcription of FAS and ME. These data support the hypothesis that glucose plays a role in mediating the effects of nutritional manipulation on transcription of FAS and ME in liver.

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Structural investigation of inhibitor designs targeting 3-dehydroquinate dehydratase from the shikimate pathway of Mycobacterium tuberculosis

Biochemical Journal ◽

10.1042/bj20110002 ◽

2011 ◽

Vol 436 (3) ◽

pp. 729-739 ◽

Cited By ~ 23

Author(s):

Marcio V. B. Dias ◽

William C. Snee ◽

Karen M. Bromfield ◽

Richard J. Payne ◽

Satheesh K. Palaninathan ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Targets ◽

Structural Investigation ◽

Catalytic Mechanism ◽

Shikimate Pathway ◽

Structural Rearrangement ◽

Competitive Inhibitors ◽

Structural Insights ◽

Potential Drug Targets

The shikimate pathway is essential in Mycobacterium tuberculosis and its absence from humans makes the enzymes of this pathway potential drug targets. In the present paper, we provide structural insights into ligand and inhibitor binding to 3-dehydroquinate dehydratase (dehydroquinase) from M. tuberculosis (MtDHQase), the third enzyme of the shikimate pathway. The enzyme has been crystallized in complex with its reaction product, 3-dehydroshikimate, and with six different competitive inhibitors. The inhibitor 2,3-anhydroquinate mimics the flattened enol/enolate reaction intermediate and serves as an anchor molecule for four of the inhibitors investigated. MtDHQase also forms a complex with citrazinic acid, a planar analogue of the reaction product. The structure of MtDHQase in complex with a 2,3-anhydroquinate moiety attached to a biaryl group shows that this group extends to an active-site subpocket inducing significant structural rearrangement. The flexible extensions of inhibitors designed to form π-stacking interactions with the catalytic Tyr24 have been investigated. The high-resolution crystal structures of the MtDHQase complexes provide structural evidence for the role of the loop residues 19–24 in MtDHQase ligand binding and catalytic mechanism and provide a rationale for the design and efficacy of inhibitors.

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Faculty Opinions recommendation of Illumination of serotonin transporter mechanism and role of the allosteric site.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.741261287.793590388 ◽

2021 ◽

Author(s):

Gary Rudnick

Keyword(s):

Serotonin Transporter ◽

Allosteric Site

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Structural insights and evaluation of the potential impact of missense variants on the interactions of SLIT2 with ROBO1/4 in cancer progression

Scientific Reports ◽

10.1038/s41598-020-78882-2 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Debmalya Sengupta ◽

Gairika Bhattacharya ◽

Sayak Ganguli ◽

Mainak Sengupta

Keyword(s):

Lung Cancer ◽

Cancer Progression ◽

Structural Parameters ◽

Homology Modelling ◽

Missense Variants ◽

Potential Impact ◽

The Impact ◽

Structural Insights ◽

Rigid Docking

AbstractThe cognate interaction of ROBO1/4 with its ligand SLIT2 is known to be involved in lung cancer progression. However, the precise role of genetic variants, disrupting the molecular interactions is less understood. All cancer-associated missense variants of ROBO1/4 and SLIT2 from COSMIC were screened for their pathogenicity. Homology modelling was done in Modeller 9.17, followed by molecular simulation in GROMACS. Rigid docking was performed for the cognate partners in PatchDock with refinement in HADDOCK server. Post-docking alterations in conformational, stoichiometric, as well as structural parameters, were assessed. The disruptive variants were ranked using a weighted scoring scheme. In silico prioritisation of 825 variants revealed 379 to be potentially pathogenic out of which, about 12% of the variants, i.e. ROBO1 (14), ROBO4 (8), and SLIT2 (23) altered the cognate docking. Six variants of ROBO1 and 5 variants of ROBO4 were identified as "high disruptors" of interactions with SLIT2 wild type. Likewise, 17 and 13 variants of SLIT2 were found to be "high disruptors" of its interaction with ROBO1 and ROBO4, respectively. Our study is the first report on the impact of cancer-associated missense variants on ROBO1/4 and SLIT2 interactions that might be the drivers of lung cancer progression.

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