Solution structure and catalytic mechanism of human protein histidine phosphatase 1

2009 ◽  
Vol 418 (2) ◽  
pp. 337-344 ◽  
Author(s):  
Weibin Gong ◽  
Yifei Li ◽  
Gaofeng Cui ◽  
Jicheng Hu ◽  
Huaming Fang ◽  
...  
Keyword(s):  
Active Site ◽  
Bound States ◽  
Solution Structure ◽  
Catalytic Mechanism ◽  
Imidazole Ring ◽  
Side Chain ◽  
Cellular Functions ◽  
Solution Structures ◽  
Histidine Phosphorylation ◽  
Mutagenesis Study

Protein histidine phosphorylation exists widely in vertebrates, and it plays important roles in signal transduction and other cellular functions. However, knowledge about eukaryotic PHPT (protein histidine phosphatase) is still very limited. To date, only one vertebrate PHPT has been discovered, and two crystal structures of hPHPT1 (human PHPT1) have been solved. However, these two structures gave different ligand-binding sites and co-ordination patterns. In the present paper, we have solved the solution structures of hPHPT1 in both Pi-free and Pi-bound states. Through comparison of the structures, along with a mutagenesis study, we have determined the active site of hPHPT1. In contrast with previous results, our results indicate that the active site is located between helix α1 and loop L5. His53 was identified to be the catalytic residue, and the NH groups of residues His53, Ala54 and Ala96 and the OH group of Ser94 should act as anchors of Pi or substrate by forming H-bonds with Pi. On the basis of our results, a catalytic mechanism is proposed for hPHPT1: the imidazole ring of His53 serves as a general base to activate a water molecule, and the activated water would attack the substrate as a nucleophile in the catalysis; the positively charged side chain of Lys21 can help stabilize the transition state. No similar catalytic mechanism can be found in the EzCatDB database.

scholarly journals X-ray structure of a putative reaction intermediate of 5-aminolaevulinic acid dehydratase

2003 ◽  
Vol 373 (3) ◽  
pp. 733-738 ◽  
Author(s):  
Peter T. ERSKINE ◽  
Leighton COATES ◽  
Danica BUTLER ◽  
James H. YOUELL ◽  
Amanda A. BRINDLEY ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Zinc Ion ◽  
Side Chain ◽  
Hydroxide Ion ◽  
X Ray ◽  
Aminolaevulinic Acid ◽  
Acid Dehydratase ◽  
Cyclic Intermediate ◽  
Aminolaevulinic Acid Dehydratase

The X-ray structure of yeast 5-aminolaevulinic acid dehydratase, in which the catalytic site of the enzyme is complexed with a putative cyclic intermediate composed of both substrate moieties, has been solved at 0.16 nm (1.6 Å) resolution. The cyclic intermediate is bound covalently to Lys263 with the amino group of the aminomethyl side chain ligated to the active-site zinc ion in a position normally occupied by a catalytic hydroxide ion. The cyclic intermediate is catalytically competent, as shown by its turnover in the presence of added substrate to form porphobilinogen. The findings, combined with those of previous studies, are consistent with a catalytic mechanism in which the C–C bond linking both substrates in the intermediate is formed before the C–N bond.

scholarly journals Laboratory variants GESG170L, GESG170K and GESG170H increase carbapenems hydrolysis and confer resistance to clavulanic acid

2021 ◽  
Author(s):  
Alessandra Piccirilli ◽  
Paola Sandra Mercuri ◽  
Bernardetta Segatore ◽  
Moreno Galleni ◽  
Fabrizia Brisdelli ◽  
...  
Keyword(s):  
Water Molecule ◽  
Clavulanic Acid ◽  
Active Site ◽  
Imidazole Ring ◽  
Hydrolytic Activity ◽  
Side Chain

The GESG170H, GESG170L and GESG170K mutants showed kcat, Km and kcat/Km values very dissimilar to GES-1 and GES-5. The enhancement of the hydrolytic activity against carbapenems is potentially due to a shift of the substrate in the active site that provides better positioning of the deacylating water molecule caused by the presence of the imidazole ring of H170 and of the long side chain of K170 and L170.

scholarly journals Solution Structure of a Low-Molecular-Weight Protein Tyrosine Phosphatase from Bacillus subtilis

2006 ◽  
Vol 188 (4) ◽  
pp. 1509-1517 ◽  
Author(s):  
Huimin Xu ◽  
Bin Xia ◽  
Changwen Jin
Keyword(s):  
Molecular Weight ◽  
Bacillus Subtilis ◽  
Active Site ◽  
Solution Structure ◽  
Catalytic Mechanism ◽  
Backbone Dynamics ◽  
Low Molecular Weight ◽  
Hydrogen Bond Interaction ◽  
Protein Tyrosine ◽  
Loop Region

ABSTRACT The low-molecular-weight (LMW) protein tyrosine phosphatases (PTPs) exist ubiquitously in prokaryotes and eukaryotes and play important roles in cellular processes. We report here the solution structure of YwlE, an LMW PTP identified from the gram-positive bacteria Bacillus subtilis. YwlE consists of a twisted central four-stranded parallel β-sheet with seven α-helices packing on both sides. Similar to LMW PTPs from other organisms, the conformation of the YwlE active site is favorable for phosphotyrosine binding, indicating that it may share a common catalytic mechanism in the hydrolysis of phosphate on tyrosine residue in proteins. Though the overall structure resembles that of the eukaryotic LMW PTPs, significant differences were observed around the active site. Residue Asp115 is likely interacting with residue Arg13 through electrostatic interaction or hydrogen bond interaction to stabilize the conformation of the active cavity, which may be a unique character of bacterial LMW PTPs. Residues in the loop region from Phe40 to Thr48 forming a wall of the active cavity are more flexible than those in other regions. Ala41 and Gly45 are located near the active cavity and form a noncharged surface around it. These unique properties demonstrate that this loop may be involved in interaction with specific substrates. In addition, the results from spin relaxation experiments elucidate further insights into the mobility of the active site. The solution structure in combination with the backbone dynamics provides insights into the mechanism of substrate specificity of bacterial LMW PTPs.

Structural and functional roles of dynamically correlated residues in thymidylate kinase

2018 ◽  
Vol 74 (4) ◽  
pp. 341-354 ◽  
Author(s):  
Santosh Kumar Chaudhary ◽  
Jeyaraman Jeyakanthan ◽  
Kanagaraj Sekar
Keyword(s):  
Molecular Dynamics ◽  
Conformational Changes ◽  
Active Site ◽  
Bound States ◽  
Catalytic Mechanism ◽  
Phosphoryl Transfer ◽  
Active Site Residues ◽  
Thymidylate Kinase ◽  
Binary And Ternary Complexes ◽  
Insight Into

Thymidylate kinase is an important enzyme in DNA synthesis. It catalyzes the conversion of thymidine monophosphate to thymidine diphosphate, with ATP as the preferred phosphoryl donor, in the presence of Mg2+. In this study, the dynamics of the active site and the communication paths between the substrates, ATP and TMP, are reported for thymidylate kinase fromThermus thermophilus. Conformational changes upon ligand binding and the path for communication between the substrates and the protein are important in understanding the catalytic mechanism of the enzyme. High-resolution X-ray crystal structures of thymidylate kinase in apo and ligand-bound states were solved. This is the first report of structures of binary and ternary complexes of thymidylate kinase with its natural substrates ATP and ATP–TMP, respectively. Distinct conformations of the active-site residues, the P-loop and the LID region observed in the apo and ligand-bound structures revealed that their concerted motion is required for the binding and proper positioning of the substrate TMP. Structural analyses provide an insight into the mode of substrate binding at the active site. The residues involved in communication between the substrates were identified through network analysis using molecular-dynamics simulations. The residues identified showed high sequence conservation across species. Biochemical analyses show that mutations of these residues either resulted in a loss of activity or affected the thermal stability of the protein. Further, molecular-dynamics analyses of mutants suggest that the proper positioning of TMP is important for catalysis. These data also provide an insight into the phosphoryl-transfer mechanism.

scholarly journals Solution structures of the Bacillus cereus metallo-β-lactamase BcII and its complex with the broad spectrum inhibitor R-thiomandelic acid

2013 ◽  
Vol 456 (3) ◽  
pp. 397-407 ◽  
Author(s):  
Andreas Ioannis Karsisiotis ◽  
Christian F. Damblon ◽  
Gordon C. K. Roberts
Keyword(s):  
Antibiotic Resistance ◽  
Bacillus Cereus ◽  
Broad Spectrum ◽  
Active Site ◽  
Solution Structure ◽  
Inhibitor Binding ◽  
Solution Structures ◽  
Micro Organisms ◽  
Flexible Loops

Metallo-β-lactamases are important in antibiotic resistance in micro-organisms. We report the first solution structure of a metallo-β-lactamase and its complex with an inhibitor, allowing the key flexible loops flanking the active site and their role in inhibitor binding to be properly defined.

scholarly journals Determination of the ionization state of the active-site histidine in a subtilisin-(chloromethane inhibitor) derivative by 13C-NMR

1996 ◽  
Vol 317 (1) ◽  
pp. 35-40 ◽  
Author(s):  
Timothy P. O'CONNELL ◽  
J. Paul G. MALTHOUSE
Keyword(s):  
13C Nmr ◽  
Active Site ◽  
Catalytic Mechanism ◽  
13C Nmr Spectroscopy ◽  
Imidazole Ring ◽  
Histidine Residue ◽  
Ionization State ◽  
Methylene Carbon ◽  
Active Site Histidine

Subtilisin BPN´ has been alkylated using benzyloxycarbonylglycylglycyl[1-13C]phenylalanylchloromethane. Using difference 13C-NMR spectroscopy a single signal due to the 13C-enriched α-methylene carbon of the subtilisin–(chloromethane inhibitor) derivative was detected. No evidence for the denaturation/autolysis of this derivative was obtained from pH 3.5 to 11.5. However, incubating at pH 12.75 or heating in the presence of SDS at pH 6.9 did denature this derivative. The negative titration shift of the α-methylene carbon of the denatured derivatives confirmed that the inhibitor had alkylated N-3 of the imidazole ring of the active-site histidine. The positive titration shift of 3.96 p.p.m. and the pKa of 7.04 obtained from studying the native subtilisin–(chloromethane inhibitor) derivative are assigned to oxyanion formation. We conclude that the pKa of the alkylated histidine residue in the native subtilisin–(chloromethane inhibitor) derivative must be > 12 and that subtilisin preferentially stabilizes the zwitterionic tetrahedral adduct consisting of the oxyanion and the imidazolium ion of the active-site histidine residue. We show that even before the oxyanion is formed the pKa of the active-site histidine must be much greater than that of the oxyanion in the zwitterionic tetrahedral adduct. We discuss the significance of our results for the catalytic mechanism of the serine proteinases.

scholarly journals One-carbon chemistry of oxalate oxidoreductase captured by X-ray crystallography

2015 ◽  
Vol 113 (2) ◽  
pp. 320-325 ◽  
Author(s):  
Marcus I. Gibson ◽  
Percival Yang-Ting Chen ◽  
Aileen C. Johnson ◽  
Elizabeth Pierce ◽  
Mehmet Can ◽  
...  
Keyword(s):  
Active Site ◽  
Bound States ◽  
Thiamine Pyrophosphate ◽  
Side Chain ◽  
X Ray ◽  
X Ray Crystallography ◽  
Acetogenic Bacteria ◽  
Switch Mechanism ◽  
Covalent Adducts ◽  
Positively Charged

Thiamine pyrophosphate (TPP)-dependent oxalate oxidoreductase (OOR) metabolizes oxalate, generating two molecules of CO2and two low-potential electrons, thus providing both the carbon and reducing equivalents for operation of the Wood−Ljungdahl pathway of acetogenesis. Here we present structures of OOR in which two different reaction intermediate bound states have been trapped: the covalent adducts between TPP and oxalate and between TPP and CO2. These structures, along with the previously determined structure of substrate-free OOR, allow us to visualize how active site rearrangements can drive catalysis. Our results suggest that OOR operates via a bait-and-switch mechanism, attracting substrate into the active site through the presence of positively charged and polar residues, and then altering the electrostatic environment through loop and side chain movements to drive catalysis. This simple but elegant mechanism explains how oxalate, a molecule that humans and most animals cannot break down, can be used for growth by acetogenic bacteria.

Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

2019 ◽  
Vol 476 (21) ◽  
pp. 3333-3353 ◽  
Author(s):  
Malti Yadav ◽  
Kamalendu Pal ◽  
Udayaditya Sen
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Triosephosphate Isomerase ◽  
Catalytic Mechanism ◽  
Degradation Mechanism ◽  
Structural Basis ◽  
Conserved Residues ◽  
Phosphodiester Bond ◽  
Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

scholarly journals The Solution Structure of the Human ETS1-DNA Complex Reveals a Novel Mode of Binding and True Side Chain Intercalation

Cell ◽  
1996 ◽  
Vol 87 (2) ◽  
pp. 358 ◽  
Author(s):  
Milton H Werner ◽  
G.Marius Clore ◽  
Constance L Fisher ◽  
Robert J Fisher ◽  
Loc Trinh ◽  
...  
Keyword(s):  
Solution Structure ◽  
Side Chain ◽  
Dna Complex

scholarly journals Structure of Nanobody Nb23

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3567
Author(s):  
Mathias Percipalle ◽  
Yamanappa Hunashal ◽  
Jan Steyaert ◽  
Federico Fogolari ◽  
Gennaro Esposito
Keyword(s):  
Nmr Spectroscopy ◽  
Chemical Shift ◽  
Solution Structure ◽  
Chemical Shifts ◽  
Molecular Size ◽  
Side Chain ◽  
3D Nmr ◽  
Target Antigen ◽  
Internuclear Distances ◽  
2D And 3D

Background: Nanobodies, or VHHs, are derived from heavy chain-only antibodies (hcAbs) found in camelids. They overcome some of the inherent limitations of monoclonal antibodies (mAbs) and derivatives thereof, due to their smaller molecular size and higher stability, and thus present an alternative to mAbs for therapeutic use. Two nanobodies, Nb23 and Nb24, have been shown to similarly inhibit the self-aggregation of very amyloidogenic variants of β2-microglobulin. Here, the structure of Nb23 was modeled with the Chemical-Shift (CS)-Rosetta server using chemical shift assignments from nuclear magnetic resonance (NMR) spectroscopy experiments, and used as prior knowledge in PONDEROSA restrained modeling based on experimentally assessed internuclear distances. Further validation was comparatively obtained with the results of molecular dynamics trajectories calculated from the resulting best energy-minimized Nb23 conformers. Methods: 2D and 3D NMR spectroscopy experiments were carried out to determine the assignment of the backbone and side chain hydrogen, nitrogen and carbon resonances to extract chemical shifts and interproton separations for restrained modeling. Results: The solution structure of isolated Nb23 nanobody was determined. Conclusions: The structural analysis indicated that isolated Nb23 has a dynamic CDR3 loop distributed over different orientations with respect to Nb24, which could determine differences in target antigen affinity or complex lability.

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