scholarly journals Measurement of lysophospholipid acyltransferase activities using substrate competition

2014 ◽  
Vol 55 (4) ◽  
pp. 782-791 ◽  
Author(s):  
Sarah A. Martin ◽  
Miguel A. Gijón ◽  
Dennis R. Voelker ◽  
Robert C. Murphy
Keyword(s):  
Lysophospholipid Acyltransferase ◽  
Substrate Competition

scholarly journals Competing Substrates for the Bifunctional Diaminopimelic Acid Epimerase/Glutamate Racemase Modulate Peptidoglycan Synthesis in Chlamydia trachomatis

2020 ◽  
Vol 89 (1) ◽  
pp. e00401-20
Author(s):  
Raghuveer Singh ◽  
Jessica A. Slade ◽  
Mary Brockett ◽  
Daniel Mendez ◽  
George W. Liechti ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Diaminopimelic Acid ◽  
Competition Strategy ◽  
Content Type ◽  
E Coli ◽  
Glutamate Racemase ◽  
Heterologous System ◽  
Substrate Competition

ABSTRACTThe Chlamydia trachomatis genome encodes multiple bifunctional enzymes, such as DapF, which is capable of both diaminopimelic acid (DAP) epimerase and glutamate racemase activity. Our previous work demonstrated the bifunctional activity of chlamydial DapF in vitro and in a heterologous system (Escherichia coli). In the present study, we employed a substrate competition strategy to demonstrate DapFCt function in vivo in C. trachomatis. We reasoned that, because DapFCt utilizes a shared substrate-binding site for both racemase and epimerase activities, only one activity can occur at a time. Therefore, an excess of one substrate relative to another must determine which activity is favored. We show that the addition of excess l-glutamate or meso-DAP (mDAP) to C. trachomatis resulted in 90% reduction in bacterial titers, compared to untreated controls. Excess l-glutamate reduced in vivo synthesis of mDAP by C. trachomatis to undetectable levels, thus confirming that excess racemase substrate led to inhibition of DapFCt DAP epimerase activity. We previously showed that expression of dapFCt in a murI (racemase) ΔdapF (epimerase) double mutant of E. coli rescues the d-glutamate auxotrophic defect. Addition of excess mDAP inhibited growth of this strain, but overexpression of dapFCt allowed the mutant to overcome growth inhibition. These results confirm that DapFCt is the primary target of these mDAP and l-glutamate treatments. Our findings demonstrate that suppression of either the glutamate racemase or epimerase activity of DapF compromises the growth of C. trachomatis. Thus, a substrate competition strategy can be a useful tool for in vivo validation of an essential bifunctional enzyme.

scholarly journals Macrocyclic Peptides as Allosteric Inhibitors of Nicotinamide N-Methyltransferase (NNMT)

2020 ◽  
Author(s):  
Matthijs J. van Haren ◽  
Yurui Zhang ◽  
Ned Buijs ◽  
Vito Thijssen ◽  
Davide Sartini ◽  
...  
Keyword(s):  
Cyclic Peptides ◽  
Cyclic Peptide ◽  
Peptide Inhibitors ◽  
Allosteric Inhibitors ◽  
Disease States ◽  
Potent Inhibition ◽  
Macrocyclic Peptides ◽  
Diabetes And Obesity ◽  
Substrate Competition

<p>Nicotinamide <i>N</i>-methyltransferase (NNMT) methylates nicotinamide to form 1-methylnicotinamide using <i>S</i>-adenosyl-l-methionine (SAM) as the methyl donor. The complexity of the role of NNMT in healthy and disease states is slowly being elucidated and provides indication that NNMT may be an interesting therapeutic target for a variety of diseases including cancer, diabetes, and obesity. Most inhibitors of NNMT described to date are structurally related to one or both of its substrates. In search of structurally diverse NNMT inhibitors, an mRNA display screening technique was used to identify macrocyclic peptides which bind to NNMT. Several of the cyclic peptides identified in this manner show potent inhibition of NNMT with IC<sub>50</sub> values as low as 229 nM. Interestingly, substrate competition experiments reveal that these cyclic peptide inhibitors are noncompetitive with either SAM or NA indicating they may be the first allosteric inhibitors reported for NNMT.</p>

scholarly journals Gene Regulation by MAPK Substrate Competition

2011 ◽  
Vol 20 (6) ◽  
pp. 880-887 ◽  
Author(s):  
Yoosik Kim ◽  
María José Andreu ◽  
Bomyi Lim ◽  
Kwanghun Chung ◽  
Mark Terayama ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Substrate Competition

Application of anaerobic biological treatment for sulphate removal in viscose industry wastewater

2007 ◽  
Vol 55 (6) ◽  
pp. 181-189 ◽  
Author(s):  
V. Parravicini ◽  
K. Svardal ◽  
H. Kroiss
Keyword(s):  
Large Scale ◽  
Anaerobic Treatment ◽  
Methanogenic Bacteria ◽  
Fibre Industry ◽  
Process Conditions ◽  
Bench Scale ◽  
Sulphide Concentration ◽  
Major Interest ◽  
Sulphate Removal ◽  
Substrate Competition

Long term lab-scale and bench-scale experiments were performed to investigate the feasibility of the anaerobic process to treat wastewater from a pulp and viscose fibre industry. Anaerobic wastewater treatment enables an advantageous combination of COD, sulphate and zinc removal from viscose wastewater. The aim of the investigations was to evaluate the influence of the free sulphide concentration on COD and sulphate removal efficiency and on the substrate competition between sulphate reducing and methanogenic bacteria. Since the wastewater did not contain enough COD for complete sulphate removal it was of major interest to determine favourable process conditions to steer the substrate competition in favour of sulphate reduction. Further experiments at bench-scale permitted us to evaluate applicable COD-loading rates and gain fundamental information about process stability and optimization for large-scale implementation. The present work will deal with the most relevant experimental results achieved and with important technological aspects of anaerobic treatment of viscose wastewater.

scholarly journals pH- and sodium-induced changes in a sodium/proton antiporter

2014 ◽  
Vol 70 (a1) ◽  
pp. C1064-C1064
Author(s):  
Cristina Paulino ◽  
Werner Kühlbrandt
Keyword(s):  
Conformational Changes ◽  
Dissociation Constants ◽  
Major Effect ◽  
Ion Binding ◽  
Ion Translocation ◽  
Common Ion ◽  
Induced Changes ◽  
3D Em ◽  
2D Crystals ◽  
Substrate Competition

Na+/H+antiporters are essential secondary-active transporters that are found across all biological kingdoms and play a crucial role in the pH, sodium and cell volume homeostasis. MjNhaP1 is an archaeal electroneutral Na+/H+-antiporter resembling the human NHE1 exchanger. Substrate-induced conformational changes in MjNhaP1 were examined by electron crystallography of 2D crystals in a range of physiological pH and Na+conditions. In the absence of sodium, changes in pH had no major effect on the structure of MjNhaP1, whereas changes in Na+concentration caused a marked conformational change that was largely pH-independent. Crystallographically determined, apparent dissociation constants indicated ~10-fold stronger Na+binding at pH 8 than at pH 4, consistent with substrate competition for a common ion-binding site. In conjunction with a new 3D EM map of MjNhaP1 a model for transport mechanism is proposed. Conformational changes occur in the 6-helix bundle region of MjNhaP1 that is thought to harbour the ion translocation site. Na+-binding converts the antiporter from the apo- or proton-bound, outward-open state to the Na+-bound, inward-open state. Oscillation between these two states result in rapid Na+/H+antiport.

scholarly journals Substrate Competition as a Source of Ultrasensitivity in the Inactivation of Wee1

Cell ◽  
2007 ◽  
Vol 128 (6) ◽  
pp. 1133-1145 ◽  
Author(s):  
Sun Young Kim ◽  
James E. Ferrell
Keyword(s):  
Substrate Competition
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