Ligand-bound structures of 3-deoxy-D-manno-octulosonate 8-phosphate phosphatase fromMoraxella catarrhalisreveal a water channel connecting to the active site for the second step of catalysis

2015 ◽  
Vol 71 (2) ◽  
pp. 239-255 ◽  
Author(s):  
Sonali Dhindwal ◽  
Priyanka Priyadarshini ◽  
Dipak N. Patil ◽  
Satya Tapas ◽  
Pramod Kumar ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Metal Ion ◽  
Water Channel ◽  
Second Step ◽  
Phosphoryl Transfer ◽  
Water Molecules ◽  
Crystal Forms ◽  
Tetrameric Structure ◽  
Site Water

KdsC, the third enzyme of the 3-deoxy-D-manno-octulosonic acid (KDO) biosynthetic pathway, catalyzes a substrate-specific reaction to hydrolyze 3-deoxy-D-manno-octulosonate 8-phosphate to generate a molecule of KDO and phosphate. KdsC is a phosphatase that belongs to the C0 subfamily of the HAD superfamily. To understand the molecular basis for the substrate specificity of this tetrameric enzyme, the crystal structures of KdsC fromMoraxella catarrhalis(Mc-KdsC) with several combinations of ligands, namely metal ion, citrate and products, were determined. Various transition states of the enzyme have been captured in these crystal forms. The ligand-free and ligand-bound crystal forms reveal that the binding of ligands does not cause any specific conformational changes in the active site. However, the electron-density maps clearly showed that the conformation of KDO as a substrate is different from the conformation adopted by KDO when it binds as a cleaved product. Furthermore, structural evidence for the existence of an intersubunit tunnel has been reported for the first time in the C0 subfamily of enzymes. A role for this tunnel in transferring water molecules from the interior of the tetrameric structure to the active-site cleft has been proposed. At the active site, water molecules are required for the formation of a water bridge that participates as a proton shuttle during the second step of the two-step phosphoryl-transfer reaction. In addition, as the KDO biosynthesis pathway is a potential antibacterial target, pharmacophore-based virtual screening was employed to identify inhibitor molecules for theMc-KdsC enzyme.

scholarly journals Relebactam Is a Potent Inhibitor of the KPC-2 β-Lactamase and Restores Imipenem Susceptibility in KPC-Producing Enterobacteriaceae

2018 ◽  
Vol 62 (6) ◽  
Author(s):  
Krisztina M. Papp-Wallace ◽  
Melissa D. Barnes ◽  
Jim Alsop ◽  
Magdalena A. Taracila ◽  
Christopher R. Bethel ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Rate Constant ◽  
Active Site ◽  
Klebsiella Oxytoca ◽  
Enterobacter Aerogenes ◽  
Clinical Isolates ◽  
Piperidine Ring ◽  
Water Molecules ◽  
Content Type ◽  
Site Water

ABSTRACT The imipenem-relebactam combination is in development as a potential treatment regimen for infections caused by Enterobacteriaceae possessing complex β-lactamase backgrounds. Relebactam is a β-lactamase inhibitor that possesses the diazabicyclooctane core, as in avibactam; however, the R1 side chain of relebactam also includes a piperidine ring, whereas that of avibactam is a carboxyamide. Here, we investigated the inactivation of the Klebsiella pneumoniae carbapenemase KPC-2, the most widespread class A carbapenemase, by relebactam and performed susceptibility testing with imipenem-relebactam using KPC-producing clinical isolates of Enterobacteriaceae . MIC measurements using agar dilution methods revealed that all 101 clinical isolates of KPC-producing Enterobacteriaceae ( K. pneumoniae , Klebsiella oxytoca , Enterobacter cloacae , Enterobacter aerogenes , Citrobacter freundii , Citrobacter koseri , and Escherichia coli ) were highly susceptible to imipenem-relebactam (MICs ≤ 2 mg/liter). Relebactam inhibited KPC-2 with a second-order onset of acylation rate constant ( k 2 / K ) value of 24,750 M −1 s −1 and demonstrated a slow off-rate constant ( k off ) of 0.0002 s −1 . Biochemical analysis using time-based mass spectrometry to map intermediates revealed that the KPC-2–relebactam acyl-enzyme complex was stable for up to 24 h. Importantly, desulfation of relebactam was not observed using mass spectrometry. Desulfation and subsequent deacylation have been observed during the reaction of KPC-2 with avibactam. Upon molecular dynamics simulations of relebactam in the KPC-2 active site, we found that the positioning of active-site water molecules is less favorable for desulfation in the KPC-2 active site than it is in the KPC-2–avibactam complex. In the acyl complexes, the water molecules are within 2.5 to 3 Å of the avibactam sulfate; however, they are more than 5 to 6 Å from the relebactam sulfate. As a result, we propose that the KPC-2–relebactam acyl complex is more stable than the KPC-2–avibactam complex. The clinical implications of this difference are not currently known.

scholarly journals The three-dimensional structure of a class-Pi glutathione S-transferase complexed with glutathione: the active-site hydration provides insights into the reaction mechanism

1998 ◽  
Vol 333 (3) ◽  
pp. 811-816 ◽  
Author(s):  
Antonio PÁRRAGA ◽  
Isabel GARCÍA-SÁEZ ◽  
Sinead B. WALSH ◽  
Timothy J. MANTLE ◽  
Miquel COLL
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Water Molecules ◽  
X Ray Diffraction ◽  
Glutathione S Transferase ◽  
X Ray ◽  
The Right

The structure of mouse liver glutathione S-transferase P1-1 complexed with its substrate glutathione (GSH) has been determined by X-ray diffraction analysis. No conformational changes in the glutathione moiety or in the protein, other than small adjustments of some side chains, are observed when compared with glutathione adduct complexes. Our structure confirms that the role of Tyr-7 is to stabilize the thiolate by hydrogen bonding and to position it in the right orientation. A comparison of the enzyme–GSH structure reported here with previously described structures reveals rearrangements in a well-defined network of water molecules in the active site. One of these water molecules (W0), identified in the unliganded enzyme (carboxymethylated at Cys-47), is displaced by the binding of GSH, and a further water molecule (W4) is displaced following the binding of the electrophilic substrate and the formation of the glutathione conjugate. The possibility that one of these water molecules participates in the proton abstraction from the glutathione thiol is discussed.

Structure of a backtracked state reveals conformational changes similar to the state following nucleotide incorporation in human norovirus polymerase

2014 ◽  
Vol 70 (12) ◽  
pp. 3099-3109 ◽  
Author(s):  
Dmitry Zamyatkin ◽  
Chandni Rao ◽  
Elesha Hoffarth ◽  
Gabriela Jurca ◽  
Hayeong Rho ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Covalent Bond ◽  
Crystal Form ◽  
Phosphoryl Group ◽  
Nucleoside Triphosphate ◽  
Orthorhombic Crystal ◽  
Crystal Forms ◽  
Terminal Nucleotide ◽  
Thumb Domain

The RNA-dependent RNA polymerase (RdRP) from norovirus (NV) genogroup II has previously been crystallized as an apoenzyme (APO1) in multiple crystal forms, as well as as a pre-incorporation ternary complex (PRE1) bound to Mn2+, various nucleoside triphosphates and an RNA primer-template duplex in an orthorhombic crystal form. When crystallized under near-identical conditions with a slightly different RNA primer/template duplex, however, the enzyme–RNA complex forms tetragonal crystals (anisotropic data,dmin≃ 1.9 Å) containing a complex with the primer/template bound in a backtracked state (BACK1) similar to a post-incorporation complex (POST1) in a step of the enzymatic cycle immediately following nucleotidyl transfer. The BACK1 conformation shows that the terminal nucleotide of the primer binds in a manner similar to the nucleoside triphosphate seen in the PRE1 complex, even though the terminal two phosphoryl groups in the triphosphate moiety are absent and a covalent bond is present between the α-phosphoryl group of the terminal nucleotide and the 3′-oxygen of the penultimate nucleotide residue. The two manganese ions bound at the active site coordinate to conserved Asp residues and the bridging phosphoryl group of the terminal nucleotide. Surprisingly, the conformation of the thumb domain in BACK1 resembles the open APO1 state more than the closed conformation seen in PRE1. The BACK1 complex thus reveals a hybrid state in which the active site is closed while the thumb domain is open. Comparison of the APO1, PRE1 and BACK1 structures of NV polymerase helps to reveal a more complete and complex pathway of conformational changes within a single RdRP enzyme system. These conformational changes lend insight into the mechanism of RNA translocation following nucleotidyl transfer and suggest novel approaches for the development of antiviral inhibitors.

scholarly journals Dimeric structures of quinol-dependent nitric oxide reductases (qNORs) revealed by cryo–electron microscopy

2019 ◽  
Vol 5 (8) ◽  
pp. eaax1803 ◽  
Author(s):  
Chai C. Gopalasingam ◽  
Rachel M. Johnson ◽  
George N. Chiduza ◽  
Takehiko Tosha ◽  
Masaki Yamamoto ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Electron Microscopy ◽  
Cytochrome C ◽  
Active Site ◽  
Proton Transport ◽  
Water Channel ◽  
Water Molecules ◽  
Cryo Electron Microscopy ◽  
Proton Source ◽  
Key Residues

Quinol-dependent nitric oxide reductases (qNORs) are membrane-integrated, iron-containing enzymes of the denitrification pathway, which catalyze the reduction of nitric oxide (NO) to the major ozone destroying gas nitrous oxide (N2O). Cryo–electron microscopy structures of active qNOR from Alcaligenes xylosoxidans and an activity-enhancing mutant have been determined to be at local resolutions of 3.7 and 3.2 Å, respectively. They unexpectedly reveal a dimeric conformation (also confirmed for qNOR from Neisseria meningitidis) and define the active-site configuration, with a clear water channel from the cytoplasm. Structure-based mutagenesis has identified key residues involved in proton transport and substrate delivery to the active site of qNORs. The proton supply direction differs from cytochrome c–dependent NOR (cNOR), where water molecules from the cytoplasm serve as a proton source similar to those from cytochrome c oxidase.

Proton conduction in alkali metal ion-exchanged porous ionic crystals

2017 ◽  
Vol 19 (43) ◽  
pp. 29077-29083 ◽  
Author(s):  
Sayaka Uchida ◽  
Reina Hosono ◽  
Ryo Eguchi ◽  
Ryosuke Kawahara ◽  
Ryota Osuga ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Alkali Metal ◽  
Ionic Crystal ◽  
Metal Ion ◽  
Water Molecules ◽  
Ionic Crystals ◽  
High Proton Conductivity ◽  
Alkali Metal Ion ◽  
Crystal Forms ◽  
High Proton

Li+ in an alkali metal ion-exchanged porous ionic crystal forms a dense and extensive hydrogen-bonding network of water molecules with mobile protons leading to a high proton conductivity (>10−3 S cm−1).

scholarly journals Structural and biochemical characteristics of two Staphylococcus epidermidis RNase J paralogs RNase J1 and RNase J2

2020 ◽  
Vol 295 (49) ◽  
pp. 16863-16876
Author(s):  
Rishi Raj ◽  
Savitha Nadig ◽  
Twinkal Patel ◽  
Balasubramanian Gopal
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Staphylococcus Epidermidis ◽  
Metal Ion ◽  
Quaternary Structure ◽  
Structural Similarity ◽  
Endonuclease Activity ◽  
Metal Cofactor ◽  
Rna Maturation ◽  
Metal Cofactors

RNase J enzymes are metallohydrolases that are involved in RNA maturation and RNA recycling, govern gene expression in bacteria, and catalyze both exonuclease and endonuclease activity. The catalytic activity of RNase J is regulated by multiple mechanisms which include oligomerization, conformational changes to aid substrate recognition, and the metal cofactor at the active site. However, little is known of how RNase J paralogs differ in expression and activity. Here we describe structural and biochemical features of two Staphylococcus epidermidis RNase J paralogs, RNase J1 and RNase J2. RNase J1 is a homodimer with exonuclease activity aided by two metal cofactors at the active site. RNase J2, on the other hand, has endonuclease activity and one metal ion at the active site and is predominantly a monomer. We note that the expression levels of these enzymes vary across Staphylococcal strains. Together, these observations suggest that multiple interacting RNase J paralogs could provide a strategy for functional improvisation utilizing differences in intracellular concentration, quaternary structure, and distinct active site architecture despite overall structural similarity.

Free and ATP-bound structures of Ap4A hydrolase fromAquifex aeolicusV5

2010 ◽  
Vol 66 (2) ◽  
pp. 116-124 ◽  
Author(s):  
Jeyaraman Jeyakanthan ◽  
Shankar Prasad Kanaujia ◽  
Yuya Nishida ◽  
Noriko Nakagawa ◽  
Surendran Praveen ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Active Site ◽  
Three Dimensional ◽  
Protein Molecule ◽  
Water Molecules ◽  
Aquifex Aeolicus ◽  
Functional Roles ◽  
Product Molecule ◽  
Dimensional Crystal

Asymmetric diadenosine tetraphosphate (Ap4A) hydrolases degrade the metabolite Ap4A back into ATP and AMP. The three-dimensional crystal structure of Ap4A hydrolase (16 kDa) fromAquifex aeolicushas been determined in free and ATP-bound forms at 1.8 and 1.95 Å resolution, respectively. The overall three-dimensional crystal structure of the enzyme shows an αβα-sandwich architecture with a characteristic loop adjacent to the catalytic site of the protein molecule. The ATP molecule is bound in the primary active site and the adenine moiety of the nucleotide binds in a ring-stacking arrangement equivalent to that observed in the X-ray structure of Ap4A hydrolase fromCaenorhabditis elegans. Binding of ATP in the active site induces local conformational changes which may have important implications in the mechanism of substrate recognition in this class of enzymes. Furthermore, two invariant water molecules have been identified and their possible structural and/or functional roles are discussed. In addition, modelling of the substrate molecule at the primary active site of the enzyme suggests a possible path for entry and/or exit of the substrate and/or product molecule.

scholarly journals Potent Inhibitors of a Shikimate Pathway Enzyme from Mycobacterium tuberculosis

2011 ◽  
Vol 286 (18) ◽  
pp. 16197-16207 ◽  
Author(s):  
Sebastian Reichau ◽  
Wanting Jiao ◽  
Scott R. Walker ◽  
Richard D. Hutton ◽  
Edward N. Baker ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Conformational Changes ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Condensation Reaction ◽  
Shikimate Pathway ◽  
Enzyme Reaction ◽  
Multidrug Resistant ◽  
Active Site Residues ◽  
Site Water

Tuberculosis remains a serious global health threat, with the emergence of multidrug-resistant strains highlighting the urgent need for novel antituberculosis drugs. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step of the shikimate pathway for the biosynthesis of aromatic compounds. This pathway has been shown to be essential in Mycobacterium tuberculosis, the pathogen responsible for tuberculosis. DAH7PS catalyzes a condensation reaction between P-enolpyruvate and erythrose 4-phosphate to give 3-deoxy-d-arabino-heptulosonate 7-phosphate. The enzyme reaction mechanism is proposed to include a tetrahedral intermediate, which is formed by attack of an active site water on the central carbon of P-enolpyruvate during the course of the reaction. Molecular modeling of this intermediate into the active site reported in this study shows a configurational preference consistent with water attack from the re face of P-enolpyruvate. Based on this model, we designed and synthesized an inhibitor of DAH7PS that mimics this reaction intermediate. Both enantiomers of this intermediate mimic were potent inhibitors of M. tuberculosis DAH7PS, with inhibitory constants in the nanomolar range. The crystal structure of the DAH7PS-inhibitor complex was solved to 2.35 Å. Both the position of the inhibitor and the conformational changes of active site residues observed in this structure correspond closely to the predictions from the intermediate modeling. This structure also identifies a water molecule that is located in the appropriate position to attack the re face of P-enolpyruvate during the course of the reaction, allowing the catalytic mechanism for this enzyme to be clearly defined.

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