scholarly journals Characterization of the Acetate Binding Pocket in the Methanosarcina thermophila Acetate Kinase

2005 ◽  
Vol 187 (7) ◽  
pp. 2386-2394 ◽  
Author(s):  
Cheryl Ingram-Smith ◽  
Andrea Gorrell ◽  
Sarah H. Lawrence ◽  
Prabha Iyer ◽  
Kerry Smith ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Binding Pocket ◽  
Acetate Kinase ◽  
Phosphoryl Group ◽  
Acetyl Phosphate ◽  
Hydrophobic Pocket ◽  
Methanosarcina Thermophila ◽  
Methyl Group ◽  
Substrate Binding Sites

ABSTRACT Acetate kinase catalyzes the reversible magnesium-dependent synthesis of acetyl phosphate by transfer of the ATP γ-phosphoryl group to acetate. Inspection of the crystal structure of the Methanosarcina thermophila enzyme containing only ADP revealed a solvent-accessible hydrophobic pocket formed by residues Val93, Leu122, Phe179, and Pro232 in the active site cleft, which identified a potential acetate binding site. The hypothesis that this was a binding site was further supported by alignment of all acetate kinase sequences available from databases, which showed strict conservation of all four residues, and the recent crystal structure of the M. thermophila enzyme with acetate bound in this pocket. Replacement of each residue in the pocket produced variants with Km values for acetate that were 7- to 26-fold greater than that of the wild type, and perturbations of this binding pocket also altered the specificity for longer-chain carboxylic acids and acetyl phosphate. The kinetic analyses of variants combined with structural modeling indicated that the pocket has roles in binding the methyl group of acetate, influencing substrate specificity, and orienting the carboxyl group. The kinetic analyses also indicated that binding of acetyl phosphate is more dependent on interactions of the phosphate group with an unidentified residue than on interactions between the methyl group and the hydrophobic pocket. The analyses also indicated that Phe179 is essential for catalysis, possibly for domain closure. Alignments of acetate kinase, propionate kinase, and butyrate kinase sequences obtained from databases suggested that these enzymes have similar catalytic mechanisms and carboxylic acid substrate binding sites.

scholarly journals Novel Pyrophosphate-Forming Acetate Kinase from the Protist Entamoeba histolytica

2012 ◽  
Vol 11 (10) ◽  
pp. 1249-1256 ◽  
Author(s):  
Matthew L. Fowler ◽  
Cheryl Ingram-Smith ◽  
Kerry S. Smith
Keyword(s):  
Entamoeba Histolytica ◽  
Acetate Kinase ◽  
Phosphoryl Group ◽  
Phosphoryl Transfer ◽  
Acetyl Phosphate ◽  
Phosphate Binding ◽  
Methanosarcina Thermophila ◽  
Inorganic Pyrophosphate ◽  
Content Type ◽  
Cell Extracts

ABSTRACTAcetate kinase (ACK) catalyzes the reversible synthesis of acetyl phosphate by transfer of the γ-phosphate of ATP to acetate. Here we report the first biochemical and kinetic characterization of a eukaryotic ACK, that from the protistEntamoeba histolytica. Our characterization revealed that this protist ACK is the only known member of the ASKHA structural superfamily, which includes acetate kinase, hexokinase, and other sugar kinases, to utilize inorganic pyrophosphate (PPi)/inorganic phosphate (Pi) as the sole phosphoryl donor/acceptor. Detection of ACK activity inE. histolyticacell extracts in the direction of acetate/PPiformation but not in the direction of acetyl phosphate/Piformation suggests that the physiological direction of the reaction is toward acetate/PPiproduction. Kinetic parameters determined for each direction of the reaction are consistent with this observation. TheE. histolyticaPPi-forming ACK follows a sequential mechanism, supporting a direct in-line phosphoryl transfer mechanism as previously reported for the well-characterizedMethanosarcina thermophilaATP-dependent ACK. Characterizations of enzyme variants altered in the putative acetate/acetyl phosphate binding pocket suggested that acetyl phosphate binding is not mediated solely through a hydrophobic interaction but also through the phosphoryl group, as for theM. thermophilaACK. However, there are key differences in the roles of certain active site residues between the two enzymes. The absence of known ACK partner enzymes raises the possibility that ACK is part of a novel pathway inEntamoeba.

scholarly journals One Intact Transmembrane Substrate Binding Site Is Sufficient for the Function of the Homodimeric Type I ATP-Binding Cassette Importer for Positively Charged Amino Acids Art(MP) 2 of Geobacillus stearothermophilus

2018 ◽  
Vol 200 (12) ◽  
Author(s):  
Johanna Heuveling ◽  
Heidi Landmesser ◽  
Erwin Schneider
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Atp Binding ◽  
Content Type ◽  
Charged Amino Acids ◽  
Substrate Binding Sites ◽  
Positively Charged ◽  
Atp Binding Cassette

ABSTRACT ATP-binding cassette (ABC) transport systems comprise two transmembrane domains/subunits that form a translocation path and two nucleotide-binding domains/subunits that bind and hydrolyze ATP. Prokaryotic canonical ABC import systems require an extracellular substrate-binding protein for function. Knowledge of substrate-binding sites within the transmembrane subunits is scarce. Recent crystal structures of the ABC importer Art(QN) 2 for positively charged amino acids of Thermoanerobacter tengcongensis revealed the presence of one substrate molecule in a defined binding pocket in each of the transmembrane subunits, ArtQ (J. Yu, J. Ge, J. Heuveling, E. Schneider, and M. Yang, Proc Natl Acad Sci U S A 112:5243–5248, 2015, https://doi.org/10.1073/pnas.1415037112 ). This finding raised the question of whether both sites must be loaded with substrate prior to initiation of the transport cycle. To address this matter, we first explored the role of key residues that form the binding pocket in the closely related Art(MP) 2 transporter of Geobacillus stearothermophilus , by monitoring consequences of mutations in ArtM on ATPase and transport activity at the level of purified proteins embedded in liposomes. Our results emphasize that two negatively charged residues (E153 and D160) are crucial for wild-type function. Furthermore, the variant Art[M(L67D)P] 2 exhibited strongly impaired activities, which is why it was considered for construction of a hybrid complex containing one intact and one impaired substrate-binding site. Activity assays clearly revealed that one intact binding site was sufficient for function. To our knowledge, our study provides the first biochemical evidence on transmembrane substrate-binding sites of an ABC importer. IMPORTANCE Canonical prokaryotic ATP-binding cassette importers mediate the uptake of a large variety of chemicals, including nutrients, osmoprotectants, growth factors, and trace elements. Some also play a role in bacterial pathogenesis, which is why full understanding of their mode of action is of the utmost importance. One of the unsolved problems refers to the chemical nature and number of substrate binding sites formed by the transmembrane subunits. Here, we report that a hybrid amino acid transporter of G. stearothermophilus , encompassing one intact and one impaired transmembrane binding site, is fully competent in transport, suggesting that the binding of one substrate molecule is sufficient to trigger the translocation process.

scholarly journals Instability of ackA (Acetate Kinase) Mutations and Their Effects on Acetyl Phosphate and ATP Amounts in Streptococcus pneumoniae D39

2010 ◽  
Vol 192 (24) ◽  
pp. 6390-6400 ◽  
Author(s):  
Smirla Ramos-Montañez ◽  
Krystyna M. Kazmierczak ◽  
Kristy L. Hentchel ◽  
Malcolm E. Winkler
Keyword(s):  
Streptococcus Pneumoniae ◽  
Physiological Role ◽  
Microarray Experiment ◽  
Experimental Manipulation ◽  
Anaerobic Growth ◽  
Respiratory Pathogen ◽  
Acetate Kinase ◽  
Phosphoryl Group ◽  
Acetyl Phosphate ◽  
Response Regulators

ABSTRACT Acetyl phosphate (AcP) is a small-molecule metabolite that can act as a phosphoryl group donor for response regulators of two-component systems (TCSs). The serious human respiratory pathogen Streptococcus pneumoniae (pneumococcus) synthesizes AcP by the conventional pathway involving phosphotransacetylase and acetate kinase, encoded by pta and ackA, respectively. In addition, pneumococcus synthesizes copious amounts of AcP and hydrogen peroxide (H2O2) by pyruvate oxidase, which is encoded by spxB. To assess possible roles of AcP in pneumococcal TCS regulation and metabolism, we constructed strains with combinations of spxB, pta, and ackA mutations and determined their effects on ATP, AcP, and H2O2 production. Unexpectedly, ΔackA mutants were unstable and readily accumulated primary suppressor mutations in spxB or its positive regulator, spxR, thereby reducing H2O2 and AcP levels, and secondary capsule mutations in cps2E or cps2C. ΔackA ΔspxB mutants contained half the cellular amount of ATP as a ΔspxB or spxB + strain. Acetate addition and anaerobic growth experiments suggested decreased ATP, rather than increased AcP, as a reason that ΔackA mutants accumulated spxB or spxR suppressors, although experimental manipulation of the AcP amount was limited. This finding and other considerations suggest that coping with endogenously produced H2O2 may require energy. Starting with a ΔspxB mutant, we constructed Δpta, ΔackA, and Δpta ΔackA mutants. Epistasis and microarray experiment results were consistent with a role for the SpxB-Pta-AckA pathway in expression of the regulons controlled by the WalRK Spn , CiaRH Spn , and LiaSR Spn TCSs involved in sensing cell wall status. However, AcP likely does not play a physiological role in TCS sensing in S. pneumoniae.

scholarly journals Crystal structure of a human neuronal nAChR extracellular domain in pentameric assembly: Ligand-bound α2 homopentamer

2016 ◽  
Vol 113 (34) ◽  
pp. 9635-9640 ◽  
Author(s):  
Nikolaos Kouvatsos ◽  
Petros Giastas ◽  
Dafni Chroni-Tzartou ◽  
Cornelia Poulopoulou ◽  
Socrates J. Tzartos
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Structural Information ◽  
Extracellular Domain ◽  
Binding Pocket ◽  
Nachr Subunit ◽  
Homologous Proteins ◽  
High Sequence Identity ◽  
Neuronal Nachr ◽  
Low Sensitivity

In this study we report the X-ray crystal structure of the extracellular domain (ECD) of the human neuronal α2 nicotinic acetylcholine receptor (nAChR) subunit in complex with the agonist epibatidine at 3.2 Å. Interestingly, α2 was crystallized as a pentamer, revealing the intersubunit interactions in a wild type neuronal nAChR ECD and the full ligand binding pocket conferred by two adjacent α subunits. The pentameric assembly presents the conserved structural scaffold observed in homologous proteins, as well as distinctive features, providing unique structural information of the binding site between principal and complementary faces. Structure-guided mutagenesis and electrophysiological data confirmed the presence of the α2(+)/α2(−) binding site on the heteromeric low sensitivity α2β2 nAChR and validated the functional importance of specific residues in α2 and β2 nAChR subunits. Given the pathological importance of the α2 nAChR subunit and the high sequence identity with α4 (78%) and other neuronal nAChR subunits, our findings offer valuable information for modeling several nAChRs and ultimately for structure-based design of subtype specific drugs against the nAChR associated diseases.

scholarly journals Identification of the site of oxidase substrate binding inScytalidium thermophilumcatalase

2018 ◽  
Vol 74 (10) ◽  
pp. 979-985 ◽  
Author(s):  
Yonca Yuzugullu Karakus ◽  
Gunce Goc ◽  
Sinem Balci ◽  
Briony A. Yorke ◽  
Chi H. Trinh ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Kinetic Analysis ◽  
Binding Site ◽  
Active Site ◽  
Oxidase Activity ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Scytalidium Thermophilum ◽  
Lateral Channel

The catalase fromScytalidium thermophilumis a homotetramer containing a hemedin each active site. Although the enzyme has a classical monofunctional catalase fold, it also possesses oxidase activity towards a number of small organics, including catechol and phenol. In order to further investigate this, the crystal structure of the complex of the catalase with the classical catalase inhibitor 3-amino-1,2,4-triazole (3TR) was determined at 1.95 Å resolution. Surprisingly, no binding to the heme site was observed; instead, 3TR occupies a binding site corresponding to the NADPH-binding pocket in mammalian catalases at the entrance to a lateral channel leading to the heme. Kinetic analysis of site-directed mutants supports the assignment of this pocket as the binding site for oxidase substrates.

Critical residues for ligand binding in blade 2 of the propeller domain of the integrin αIIb subunit

2004 ◽  
Vol 91 (01) ◽  
pp. 111-118 ◽  
Author(s):  
Tatsushiro Tamura ◽  
Jun Yamanouchi ◽  
Shigeru Fujita ◽  
Takaaki Hato
Keyword(s):  
Crystal Structure ◽  
Ligand Binding ◽  
Binding Site ◽  
Thrombus Formation ◽  
Direct Interaction ◽  
Binding Pocket ◽  
Crystal Structure Analysis ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Critical Residues

SummaryLigand binding to integrin αIIbβ3 is a key event of thrombus formation. The propeller domain of the αIIb subunit has been implicated in ligand binding. Recently, the ligand binding site of the αV propeller was determined by crystal structure analysis. However, the structural basis of ligand recognition by the αIIb propeller remains to be determined. In this study, we conducted site-directed mutagenesis of all residues located in the loops extending above blades 2 and 4 of the αIIb propeller, which are spatially close to, but distinct from, the loops that contain the binding site for an RGD ligand in the crystal structure of the αV propeller. Replacement by alanine of Q111, H112 or N114 in the loop within the blade 2 (the W2:2-3 loop in the propeller model) abolished binding of a ligand-mimetic antibody and fibrinogen to αIIbβ3 induced by different types of integrin activation including activation of αIIbβ3 by β3 cytoplasmic mutation. CHO cells stably expressing recombinant αIIbβ3 bearing Q111A, H112A or N114A mutation did not exhibit αIIbβ3mediated adhesion to fibrinogen. According to the crystal structure of αVβ3, the αV residue corresponding to αIIbN114 is exposed on the integrin surface and close to the RGD binding site. These results suggest that the Q111, H112 and N114 residues in the loop within blade 2 of the αIIb propeller are critical for ligand binding, possibly because of direct interaction with ligands or modulation of the RGD binding pocket.

scholarly journals Comparison of binding interactions of dibromoflavonoids with transthyretin.

2001 ◽  
Vol 48 (4) ◽  
pp. 885-892 ◽  
Author(s):  
T Muzioł ◽  
V Cody ◽  
A Wojtczak
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Competitive Binding ◽  
Hydroxyl Group ◽  
Side Chain ◽  
Strong Interactions ◽  
Binding Interactions ◽  
Methyl Group ◽  
Channel Entrance ◽  
Molecular Architectures

The crystal structure of rat transthyretin (rTTR) complex with the dibromoflavone EMD21388 was determined to 2.3 A resolution and refined to R = 0.203 and Rfree = 0.288. Two different orientations of EMD21388, which differ in the channel penetration by 1.6 A, were found in the A/C binding site of rTTR. The single ligand position observed in the BID site is intermediate between the two positions found in the A/C site. The position of the dibromoflavone in the B/D site is similar to that reported for dibromoaurone in human TTR. The bromine atoms of EMD21388 form strong interactions in the P3 and P3' pockets of rTTR. Due to the different molecular architectures of both ligands, dibromoflavone forms only one interaction with Lys-15 near the channel entrance, while direct interactions with the pair of Lys-15 were reported for dibromoaurone. The C3* methyl group of EMD21388 mediates the bridging interactions between two TTR subunits in the P2 pockets. The interactions of the O2* hydroxyl group of dibromoaurone with the Thr-119 side chain in the P3 pockets are not matched by similar interactions in EMD21388. Both these alternative interactions can explain the competitive binding of 3',5'-dibromoflavonoids to transthyretin.

scholarly journals Characterization of the Acetate Binding Pocket in the Methanosarcina thermophila Acetate Kinase

2005 ◽  
Vol 187 (14) ◽  
pp. 5059-5059 ◽  
Author(s):  
Cheryl Ingram-Smith ◽  
Andrea Gorrell ◽  
Sarah H. Lawrence ◽  
Prabha Iyer ◽  
Kerry Smith ◽  
...  
Keyword(s):  
Binding Pocket ◽  
Acetate Kinase ◽  
Methanosarcina Thermophila

scholarly journals Identification of Essential Glutamates in the Acetate Kinase from Methanosarcina thermophila

1998 ◽  
Vol 180 (5) ◽  
pp. 1129-1134 ◽  
Author(s):  
Kavita Singh-Wissmann ◽  
Cheryl Ingram-Smith ◽  
Rebecca D. Miles ◽  
James G. Ferry
Keyword(s):  
Active Site ◽  
Kinase Activity ◽  
Amino Acid Sequences ◽  
Acetate Kinase ◽  
Acetyl Phosphate ◽  
Methanosarcina Thermophila ◽  
High Identity ◽  
E Coli ◽  
Metal Affinity ◽  
Enzyme Intermediate

ABSTRACT Acetate kinase catalyzes the reversible phosphorylation of acetate (CH3COO− + ATP⇄CH3CO2PO3 2− + ADP). A mechanism which involves a covalent phosphoryl-enzyme intermediate has been proposed, and chemical modification studies of the enzyme from Escherichia coli indicate an unspecified glutamate residue is phosphorylated (J. A. Todhunter and D. L. Purich, Biochem. Biophys. Res. Commun. 60:273–280, 1974). Alignment of the amino acid sequences for the acetate kinases from E. coli (Bacteria domain), Methanosarcina thermophila (Archaea domain), and four other phylogenetically divergent microbes revealed high identity which included five glutamates. These glutamates were replaced in theM. thermophila enzyme to determine if any are essential for catalysis. The histidine-tagged altered enzymes were produced inE. coli and purified to electrophoretic homogeneity by metal affinity chromatography. Replacements of E384 resulted in either undetectable or extremely low kinase activity, suggesting E384 is essential for catalysis which supports the proposed mechanism. Replacement of E385 influenced the Km values for acetate and ATP with only moderate decreases ink cat, which suggests that this residue is involved in substrate binding but not catalysis. The unaltered acetate kinase was not inactivated by N-ethylmaleimide; however, replacement of E385 with cysteine conferred sensitivity toN-ethylmaleimide which was prevented by preincubation with acetate, acetyl phosphate, ATP, or ADP, suggesting that E385 is located near the active site. Replacement of E97 decreased theKm value for acetate but not ATP, suggesting this residue is involved in binding acetate. Replacement of either E32 or E334 had no significant effects on the kinetic constants, which indicates that neither residue is essential for catalysis or significantly influences the binding of acetate or ATP.

scholarly journals Prediction and Validation of a Druggable Site on Virulence Factor of Drug Resistant Burkholderia Cenocepacia

2020 ◽  
Author(s):  
Kanhaya Lal ◽  
Rafael Bermo ◽  
Jonathan Cramer ◽  
Francesca Vasile ◽  
Beat Ernst ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Virtual Screening ◽  
Binding Site ◽  
Virulence Factor ◽  
Opportunistic Pathogen ◽  
Binding Pocket ◽  
Burkholderia Cenocepacia ◽  
Drug Resistant ◽  
Novel Approach ◽  
Virtual Screening Approach

<br><p> </p> <p>We use a virtual screening approach to explore the binding pocket close to the fucose binding site in a lectin from opportunistic pathogen. This is a novel approach for designing anti-adhesive drugs, and it has been very successful since we were able to obtain leads with sub millimolar affinity. Furthermore, the crystal structure of the target protein complexed with the fragment validated the existence of this secondary binding site, opening route for new design of inhibitors</p>

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