scholarly journals Allosteric cooperation in β-lactam binding to a non-classical transpeptidase

2021 ◽  
Author(s):  
Nazia Ahmad ◽  
Sangita Kachhap ◽  
Varsha Chauhan ◽  
Pallavi Juneja ◽  
Kunal Sharma ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Catalytic Site ◽  
Allosteric Site ◽  
Sugar Moiety ◽  
Peptide Moiety ◽  
Novel Approaches ◽  
Enzyme Class

Mycobacterium tuberculosis peptidoglycan (PG) is atypical as its synthesis involves a new enzyme class, L,D-transpeptidases. Prior studies of L,D-transpeptidases have identified only the catalytic site that binds to peptide moiety of the PG substrate or β-lactam antibiotics. This insight was leveraged to develop mechanism of its activity and inhibition by β-lactams. Here we report identification of an allosteric site at a distance of 21 Å from the catalytic site that binds the sugar moiety of PG substrates (hereafter referred to as the S-pocket). This site also binds a second β-lactam molecule and influences binding at the catalytic site. We provide evidence that two β-lactam molecules bind co-operatively to this enzyme, one non covalently at the S-site and one covalently at the catalytic site. This dual β-lactam binding phenomenon is previously unknown and is an observation that may offer novel approaches for the structure-based design of new β-lactam antibiotics for M. tuberculosis.

scholarly journals Allosteric regulation of menaquinone (vitamin K2) biosynthesis in the human pathogen Mycobacterium tuberculosis

2020 ◽  
Vol 295 (12) ◽  
pp. 3759-3770 ◽  
Author(s):  
Ghader Bashiri ◽  
Laura V. Nigon ◽  
Ehab N. M. Jirgis ◽  
Ngoc Anh Thu Ho ◽  
Tamsyn Stanborough ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Active Site ◽  
Feedback Inhibition ◽  
Feedback Regulation ◽  
Signal Propagation ◽  
Biosynthesis Pathway ◽  
Human Pathogen ◽  
Energy Status ◽  
Allosteric Site ◽  
Arginine Residues

Menaquinone (vitamin K2) plays a vital role in energy generation and environmental adaptation in many bacteria, including the human pathogen Mycobacterium tuberculosis (Mtb). Although menaquinone levels are known to be tightly linked to the cellular redox/energy status of the cell, the regulatory mechanisms underpinning this phenomenon are unclear. The first committed step in menaquinone biosynthesis is catalyzed by MenD, a thiamine diphosphate–dependent enzyme comprising three domains. Domains I and III form the MenD active site, but no function has yet been ascribed to domain II. Here, we show that the last cytosolic metabolite in the menaquinone biosynthesis pathway, 1,4-dihydroxy-2-naphthoic acid (DHNA), binds to domain II of Mtb-MenD and inhibits its activity. Using X-ray crystallography of four apo- and cofactor-bound Mtb-MenD structures, along with several spectroscopy assays, we identified three arginine residues (Arg-97, Arg-277, and Arg-303) that are important for both enzyme activity and the feedback inhibition by DHNA. Among these residues, Arg-277 appeared to be particularly important for signal propagation from the allosteric site to the active site. This is the first evidence of feedback regulation of the menaquinone biosynthesis pathway in bacteria, identifying a protein-level regulatory mechanism that controls menaquinone levels within the cell and may therefore represent a good target for disrupting menaquinone biosynthesis in M. tuberculosis.

scholarly journals Inhibition patterns of a model complex mimicking the reductive half-reaction of sulphite oxidase

1996 ◽  
Vol 319 (3) ◽  
pp. 953-959 ◽  
Author(s):  
Pradeep K CHAUDHURY ◽  
Samar K DAS ◽  
Sabyasachi SARKAR
Keyword(s):  
Steady State ◽  
Ternary Complex ◽  
Mixed Type ◽  
Catalytic Site ◽  
Binary Complex ◽  
Allosteric Site ◽  
Equilibrium Conditions ◽  
Enzyme Substrate ◽  
Sulphite Oxidase ◽  
Model Complex

Different inhibition types of the saturation kinetics involving a synthesized model complex, [Bu4N]2[MoVIO2(mnt)2] (E) (where mnt2- = 1,2-dicyanoethylenedithiolate), and HSO3- as the substrate (S) by structurally similar anions SO42-, H2PO4- and H2PO3- have been shown for the first time in relevance to the reductive half reaction of the native enzyme sulphite oxidase. SO42- acts as a competitive inhibitor. The mixed-type non-competitive inhibition by H2PO4- and the sigmoidal-type inhibition by H2 PO3- are explained by a diamond-configuration random-order model. This involves a random binding sequence of the substrate and the inhibitor, and forms, in addition to two binary complexes [enzyme-substrate (ES) and enzyme-inhibitor (EI)], one enzyme-substrate-inhibitor-type ternary complex (ESI) by participation of at least one more binding site in addition to the catalytic site. This is possible in the present case only by co-ordination enhancement of molybdenum in E. This co-ordination expansion is brought about by nucleophilic attack of the substrate or the inhibitor at the molybdenum, forming a hepta-coordinated binary complex with the generation of an oxoanionic functional site, called the allosteric site. Analysis of the experimental data suggests that the inhibition by H2PO4- is due to the mechanism following either equilibrium conditions or a combination of steady-state and equilibrium conditions. With H2PO3-, the inhibition is due to the mechanism following the steady-state conditions. It is also shown that the ternary complex involving the enzyme, substrate and H2PO4- or H2 PO3- is productive, but at a lower rate than that of the enzyme-substrate binary complex. Mixed-type inhibition with H2PO4- is actually of the type called ‘partially mixed competitive and non-competitive’ as the inhibitor binds both to the catalytic site and to the allosteric site. The sigmoidal-type inhibition by H2PO3- is similar to heterotropic allosteric effect of mixed V,K type with the distinction, however, that the significance of co-operativity in this case is of kinetic importance only. Received 3 January 1996/20 May 1996; accepted 25 June 1996

scholarly journals A recombinant human ‘mini’-hexokinase is catalytically active and regulated by hexose 6-phosphates

1992 ◽  
Vol 285 (1) ◽  
pp. 193-199 ◽  
Author(s):  
M Magnani ◽  
M Bianchi ◽  
A Casabianca ◽  
V Stocchi ◽  
A Daniele ◽  
...  
Keyword(s):  
Gene Duplication ◽  
Active Form ◽  
Evolutionary Relationship ◽  
Product Inhibition ◽  
Catalytic Site ◽  
Type I ◽  
Regulatory Site ◽  
Allosteric Site ◽  
Reading Frame ◽  
Catalytically Active

Mammalian hexokinase type I is a 100 kDa enzyme that has been considered to be evolved from an ancestral 50 kDa yeast-type hexokinase, insensitive to product inhibition, by gene duplication and fusion. According to this model, and based on many experimental data, the catalytic site is associated with the C-terminal half of the enzyme, although an allosteric site for the binding of glucose 6-phosphate could be present on the N-terminal half of the molecule. We have isolated a cDNA clone of hexokinase from a lambda gt11 human placenta library comprising 2658 bp, containing a single open reading frame of 1893 nucleotides, which encodes a truncate form of hexokinase starting from asparagine-287 to the terminal serine-917. This clone was further digested with restriction enzyme NcoI to obtain almost only the C-terminal half of human hexokinase starting from methionine-455 to the terminal amino acid and was overexpressed in active form in Escherichia coli and purified by ion-exchange h.p.l.c. The overexpressed ‘mini’-hexokinase was found not only to catalyse glucose phosphorylation, but also to be inhibited by glucose 6-phosphate and other mono- and bis-phosphate sugars exactly like the complete mammalian enzyme. These results suggest that the C-terminal half of human hexokinase, in addition to the catalytic site, also contains the regulatory site and that the evolutionary relationship between the hexokinases should be reconsidered by including the appearance of a regulatory site before the gene duplication.

scholarly journals Inhibiting Mycobacterium tuberculosis CoaBC by targeting an allosteric site

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Vitor Mendes ◽  
Simon R. Green ◽  
Joanna C. Evans ◽  
Jeannine Hess ◽  
Michal Blaszczyk ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Drug Target ◽  
Biosynthetic Pathway ◽  
Model Organism ◽  
Allosteric Site ◽  
Cellular Processes ◽  
Coa Thioesters ◽  
Multiple Pathogens ◽  
Substantial Interest

AbstractCoenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic allosteric site within CoaB.

scholarly journals The Structural Basis for SARM1 Inhibition, and Activation Under Energetic Stress

2020 ◽  
Author(s):  
Michael Sporny ◽  
Julia Guez-Haddad ◽  
Tami Khazma ◽  
Avraham Yaron ◽  
Moshe Dessau ◽  
...  
Keyword(s):  
Cell Death ◽  
Catalytic Site ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Allosteric Site ◽  
Age Related ◽  
Peripheral Ring ◽  
Inhibitory Site ◽  
Cellular Metabolite ◽  
Tir Domains

AbstractSARM1 is a central executor of axonal degeneration (1). Mechanistically, SARM1 contains NADase activity, which, in response to nerve injury, depletes the key cellular metabolite, NAD+ (2–5). Interestingly, SARM1 knockout mouse models do not present any apparent physiological impairment. Yet, the lack of SARM1 protects against various neuropathies (6, 7), thereby highlighting SARM1 as a likely safe and effective drug target (8). However, the absence of a SARM1 structure, in its active or inhibited form, makes it impossible to understand the molecular basis of SARM1 inhibition, and its activation under stress conditions. In this study we present two cryo-EM maps of SARM1 (at 2.6 Å and 2.9 Å resolution). We show that the inhibited SARM1 homo-octamer assumes a packed conformation with well-ordered inner and peripheral rings. Here the catalytic TIR domains are held apart from each other and are unable to form dimers, which is a prerequisite for NADase activity. More importantly, after screening several cellular metabolites we discovered that the inactive conformation is stabilized by the binding of SARM1’s own substrate: NAD+. The NAD+ inhibitory allosteric site is located away from the NAD+ catalytic site of the TIR domain. Site-directed mutagenesis of the allosteric site leads to constitutive active SARM1. Based on our data we propose that a reduction of cellular NAD+ concentrations (an early indication of disease-associated and age-related neurodegeneration (9)) disassemble SARM1’s peripheral ring, which allows NADase activity. This leads to an energetic catastrophe and eventually cell death. The discovery of the allosteric inhibitory site opens the door for the development of effective drugs that will prevent SARM1 activation, rather than compete for binding to the NADase catalytic site.Brief descriptionIt is not known how NAD+ depletion brings about neurodegeneration. Here, we show that the intrinsic NADase activity of SARM1 is allosterically inhibited by physiological concentrations of NAD+. NAD+ stabilizes a compact, auto-inhibited conformation of the SARM1 octamer. Once NAD+ levels are depleted, the allosteric inhibition is released, enabling SARM1’s NADase activity, which eventually leads to energetic catastrophe and cell death.

scholarly journals Subunit Interactions and Composition of the Fructose 6-Phosphate Catalytic Site and the Fructose 2,6-Bisphosphate Allosteric Site of Mammalian Phosphofructokinase

2009 ◽  
Vol 284 (14) ◽  
pp. 9124-9131 ◽  
Author(s):  
Cristina Ferreras ◽  
Eloy D. Hernández ◽  
Oscar H. Martínez-Costa ◽  
Juan J. Aragón
Keyword(s):  
Catalytic Site ◽  
Allosteric Site ◽  
Subunit Interactions

scholarly journals Inhibiting Mycobacterium tuberculosis CoaBC by targeting a new allosteric site

2019 ◽  
Author(s):  
Vitor Mendes ◽  
Simon R. Green ◽  
Joanna C. Evans ◽  
Jeannine Hess ◽  
Michal Blaszczyk ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Drug Target ◽  
Biosynthetic Pathway ◽  
Model Organism ◽  
Allosteric Site ◽  
Cellular Processes ◽  
Coa Thioesters ◽  
Multiple Pathogens ◽  
Substantial Interest

AbstractCoenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent inhibitors of M. tuberculosis CoaB, which we show to bind to a novel cryptic allosteric site within CoaB.

scholarly journals Mycobacterium tuberculosis Lacking All Mycolic Acid Cyclopropanation Is Viable but Highly Attenuated and Hyperinflammatory in Mice

2012 ◽  
Vol 80 (6) ◽  
pp. 1958-1968 ◽  
Author(s):  
Daniel Barkan ◽  
Dorsaf Hedhli ◽  
Han-Guang Yan ◽  
Kris Huygen ◽  
Michael S. Glickman
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycolic Acid ◽  
Acid Modification ◽  
Content Type ◽  
Mycolic Acids ◽  
Trehalose Dimycolate ◽  
Cord Factor ◽  
Aerosol Infection ◽  
Enzyme Class

ABSTRACTMycolic acids, the major lipid of theMycobacterium tuberculosiscell wall, are modified by cyclopropane rings, methyl branches, and oxygenation through the action of eightS-adenosylmethionine (SAM)-dependent mycolic acid methyltransferases (MAMTs), encoded at four genetic loci. Mycolic acid modification has been shown to be important forM. tuberculosispathogenesis, in part through effects on the inflammatory activity of trehalose dimycolate (cord factor). Studies using the MAMT inhibitor dioctylamine have suggested that the MAMT enzyme class is essential forM. tuberculosisviability. However, it is unknown whether a cyclopropane-deficient strain ofM. tuberculosiswould be viable and what the effect of cyclopropane deficiency on virulence would be. We addressed these questions by creating and characterizingM. tuberculosisstrains lacking all functional MAMTs. Our results show thatM. tuberculosisis viable either without cyclopropanation or without cyclopropanation and any oxygenated mycolates. Characterization of these strains revealed that MAMTs are required for acid fastness and resistance to detergent stress. Complete lack of cyclopropanation confers severe attenuation during the first week after aerosol infection of the mouse, whereas complete loss of MAMTs confers attenuation in the second week of infection. Characterization of immune responses to the cyclopropane- and MAMT-deficient strains indicated that the net effect of mycolate cyclopropanation is to dampen host immunity. Taken together, our findings establish the immunomodulatory function of the mycolic acid modification pathway in pathogenesis and buttress this enzyme class as an attractive target for antimycobacterial drug development.

Sign in / Sign up

Export Citation Format

Share Document