Quaternary Structure and Catalytic Activity of theEscherichia coliRibonuclease E Amino-Terminal Catalytic Domain†

Biochemistry ◽  
2003 ◽  
Vol 42 (47) ◽  
pp. 13848-13855 ◽  
Author(s):  
Anastasia J. Callaghan ◽  
J. Günter Grossmann ◽  
Yulia U. Redko ◽  
Leopold L. Ilag ◽  
Martin C. Moncrieffe ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Quaternary Structure ◽  
Catalytic Domain ◽  
Amino Terminal

Characterization of Recombinant Human Coagulation Factor XFriuli

1996 ◽  
Vol 75 (02) ◽  
pp. 313-317 ◽  
Author(s):  
D J Kim ◽  
A Girolami ◽  
H L James
Keyword(s):  
Catalytic Domain ◽  
Coagulation Factor ◽  
Molecular Size ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Bleeding Tendency ◽  
Factor X ◽  
Amino Terminal ◽  
Normal Plasma ◽  
Plasma Factor

SummaryNaturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational y-carboxy-glutamic acid (Gla) and (β-hydroxy aspartic acid (β-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels. The specific activities of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular modeling suggested the involvement of a new H-bond between the side-chains of Ser-343 and Thr-318 as they occur in anti-parallel (3-pleated sheets near the substrate-binding pocket of pFXFr. These results support the conclusion that the observed mutation in pFXFr is responsible for its dysfunctional activation and catalytic potentials, and that it accounts for the moderate bleeding tendency in the homozygous individuals who possess this variant procoagulant.

The first crystal structure of the peptidase domain of the U32 peptidase family

2015 ◽  
Vol 71 (12) ◽  
pp. 2505-2512 ◽  
Author(s):  
Magdalena Schacherl ◽  
Angelika A. M. Montada ◽  
Elena Brunstein ◽  
Ulrich Baumann
Keyword(s):  
Crystal Structure ◽  
Catalytic Mechanism ◽  
Quaternary Structure ◽  
Catalytic Domain ◽  
Three Dimensional ◽  
Zinc Ion ◽  
Crystal Structure Analysis ◽  
Dimensional Structure ◽  
Sequence Motifs ◽  
Conserved Sequence

The U32 family is a collection of over 2500 annotated peptidases in the MEROPS database with unknown catalytic mechanism. They mainly occur in bacteria and archaea, but a few representatives have also been identified in eukarya. Many of the U32 members have been linked to pathogenicity, such as proteins fromHelicobacterandSalmonella. The first crystal structure analysis of a U32 catalytic domain fromMethanopyrus kandleri(genemk0906) reveals a modified (βα)8TIM-barrel fold with some unique features. The connecting segment between strands β7 and β8 is extended and helix α7 is located on top of the C-terminal end of the barrel body. The protein exhibits a dimeric quaternary structure in which a zinc ion is symmetrically bound by histidine and cysteine side chains from both monomers. These residues reside in conserved sequence motifs. No typical proteolytic motifs are discernible in the three-dimensional structure, and biochemical assays failed to demonstrate proteolytic activity. A tunnel in which an acetate ion is bound is located in the C-terminal part of the β-barrel. Two hydrophobic grooves lead to a tunnel at the C-terminal end of the barrel in which an acetate ion is bound. One of the grooves binds to aStrep-Tag II of another dimer in the crystal lattice. Thus, these grooves may be binding sites for hydrophobic peptides or other ligands.

scholarly journals Functional CBS modules make IMPDHs octameric

2014 ◽  
Vol 70 (a1) ◽  
pp. C1283-C1283
Author(s):  
Gilles Labesse ◽  
Thomas Alexandre ◽  
Laurène Vaupré ◽  
Isabelle Salard-Arnaud ◽  
Joséphine Lai Kee Him ◽  
...  
Keyword(s):  
Analytical Ultracentrifugation ◽  
Quaternary Structure ◽  
Catalytic Domain ◽  
Structural Data ◽  
Site Directed Mutagenesis ◽  
X Ray Crystallography ◽  
Binding Pockets ◽  
Essential Enzyme ◽  
Cbs Domains

Inosine-5'-monophosphate dehydrogenase (1, 2) (IMPDH) is a major target for antiviral, antiparasitic, antileukemic and immunosuppressive therapies. It is an ubiquitous and essential enzyme for the biosynthesis of guanosine nucleotides. Up to now, IMPDHs have been reported as tetrameric enzymes harbouring a catalytic domain and a tandem of cystathionine-ß-synthase (CBS) modules. The latter had no precise function assigned despite their nearly absolute conservation among IMPDHs and consistent indication of their importance in vivo. The aim of our study was to provide evidence for the role of the CBS modules on the quaternary structure and on the regulation of IMPDHs. A multidisciplinary approach involving enzymology, site-directed mutagenesis, analytical ultracentrifugation, X-ray crystallography, SAXS, cryo-electron microscopy and molecular modelling allowed us to demonstrate that the Pseudomonas aeruginosa IMPDH is functionally active as an octamer and allosterically regulated by MgATP via each CBS module. Revisiting deposited structural data, we found this newly discovered octameric organization conserved in other IMPDH structures. Meanwhile, we demonstrated that the human IMPDH1 formed two distinct octamers that can pile up into isolated fibres in the presence of MgATP while its pathogenic mutant D226N, localised into the CBS domains, appeared to form massively aggregating fibres. The dramatic impact of this mutation could explain the severe retinopathy adRP10. Our data (3) revealed for the first time that IMPDH has an octameric architecture modulated by MgATP binding to the CBS modules, inducing large structural rearrangements. Thus, the regulatory CBS modules in IMPDHs are functional and they can either modulate catalysis or/and macromolecular assembly. Targeting the conserved effector binding pockets identified within the CBS modules might be promising to develop antibacterial compounds or drugs to counter retinopathy onset.

The SIT4 protein phosphatase functions in late G1 for progression into S phase

1991 ◽  
Vol 11 (4) ◽  
pp. 2133-2148
Author(s):  
A Sutton ◽  
D Immanuel ◽  
K T Arndt
Keyword(s):  
Cell Cycle ◽  
Protein Phosphatase ◽  
Catalytic Domain ◽  
Function Analysis ◽  
S Phase ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Amino Terminal ◽  
Polymorphic Gene ◽  
Cycle Dependent

Saccharomyces cerevisiae strains containing temperature-sensitive mutations in the SIT4 protein phosphatase arrest in late G1 at the nonpermissive temperature. Order-of-function analysis shows that SIT4 is required in late G1 for progression into S phase. While the levels of SIT4 do not change in the cell cycle, SIT4 associates with two high-molecular-weight phosphoproteins in a cell-cycle-dependent fashion. In addition, we have identified a polymorphic gene, SSD1, that in some versions can suppress the lethality due to a deletion of SIT4 and can also partially suppress the phenotypic defects due to a null mutation in BCY1. The SSD1 protein is implicated in G1 control and has a region of similarity to the dis3 protein of Schizosaccharomyces pombe. We have also identified a gene, PPH2alpha, that in high copy number can partially suppress the growth defect of sit4 strains. The PPH2 alpha gene encodes a predicted protein that is 80% identical to the catalytic domain of mammalian type 2A protein phosphatases but also has an acidic amino-terminal extension not present in other phosphatases.

scholarly journals Functional characterization of the non-catalytic ectodomains of the nucleotide pyrophosphatase/phosphodiesterase NPP1

2003 ◽  
Vol 371 (2) ◽  
pp. 321-330 ◽  
Author(s):  
Rik GIJSBERS ◽  
Hugo CEULEMANS ◽  
Mathieu BOLLEN
Keyword(s):  
Catalytic Activity ◽  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Functional Characterization ◽  
Structure Stability ◽  
Subunit Structure ◽  
Terminal Domain ◽  
Nucleotide Pyrophosphatase ◽  
Domain Truncation ◽  
And Function

The ubiquitous nucleotide pyrophosphatases/phosphodiesterases NPP1–3 consist of a short intracellular N-terminal domain, a single transmembrane domain and a large extracellular part, comprising two somatomedin-B-like domains, a catalytic domain and a poorly defined C-terminal domain. We show here that the C-terminal domain of NPP1–3 is structurally related to a family of DNA/RNA non-specific endonucleases. However, none of the residues that are essential for catalysis by the endonucleases are conserved in NPP1–NPP3, suggesting that the nuclease-like domain of NPP1–3 does not represent a second catalytic domain. Truncation analysis revealed that the nuclease-like domain of NPP1 is required for protein stability, for the targeting of NPP1 to the plasma membrane and for the expression of catalytic activity. We also demonstrate that 16 conserved cysteines in the somatomedin-B-like domains of NPP1, in concert with two flanking cysteines, mediate the dimerization of NPP1. The K173Q polymorphism of NPP1, which maps to the second somatomedin-B-like domain and has been associated with the aetiology of insulin resistance, did not affect the dimerization or catalytic activity of NPP1, and did not endow NPP1 with an affinity for the insulin receptor. Our data suggest that the non-catalytic ectodomains contribute to the subunit structure, stability and function of NPP1–3.

scholarly journals Identification of three critical acidic residues of poly(ADP-ribose) glycohydrolase involved in catalysis: determining the PARG catalytic domain

2005 ◽  
Vol 388 (2) ◽  
pp. 493-500 ◽  
Author(s):  
Chandra N. PATEL ◽  
David W. KOH ◽  
Myron K. JACOBSON ◽  
Marcos A. OLIVEIRA
Keyword(s):  
Catalytic Activity ◽  
Catalytic Domain ◽  
Functional Characterization ◽  
Sequence Alignments ◽  
Inhibitor Binding ◽  
Conserved Sequence ◽  
Binding Residue ◽  
Acidic Residues ◽  
Hydrolysis Of

PARG [poly(ADP-ribose) glycohydrolase] catalyses the hydrolysis of α(1″→2′) or α(1‴→2″) O-glycosidic linkages of ADP-ribose polymers to produce free ADP-ribose. We investigated possible mechanistic similarities between PARG and glycosidases, which also cleave O-glycosidic linkages. Glycosidases typically utilize two acidic residues for catalysis, thus we targeted acidic residues within a conserved region of bovine PARG that has been shown to contain an inhibitor-binding site. The targeted glutamate and aspartate residues were changed to asparagine in order to minimize structural alterations. Mutants were purified and assayed for catalytic activity, as well as binding, to an immobilized PARG inhibitor to determine ability to recognize substrate. Our investigation revealed residues essential for PARG catalytic activity. Two adjacent glutamic acid residues are found in the conserved sequence Gln755-Glu-Glu757, and a third residue found in the conserved sequence Val737-Asp-Phe-Ala-Asn741. Our functional characterization of PARG residues, along with recent identification of an inhibitor-binding residue Tyr796 and a glycine-rich region Gly745-Gly-Gly747 important for PARG function, allowed us to define a PARG ‘signature sequence’ [vDFA-X3-GGg-X6–8-vQEEIRF-X3-PE-X14-E-X12-YTGYa], which we used to identify putative PARG sequences across a range of organisms. Sequence alignments, along with our mapping of PARG functional residues, suggest the presence of a conserved catalytic domain of approx. 185 residues which spans residues 610–795 in bovine PARG.

scholarly journals Plasma membrane-targeted ras GTPase-activating protein is a potent suppressor of p21ras function.

1993 ◽  
Vol 13 (4) ◽  
pp. 2420-2431 ◽  
Author(s):  
D C Huang ◽  
C J Marshall ◽  
J F Hancock
Keyword(s):  
Plasma Membrane ◽  
Catalytic Activity ◽  
Catalytic Domain ◽  
Cell Transformation ◽  
Negative Regulator ◽  
Membrane Localization ◽  
Plasma Membrane Localization ◽  
Terminal Domain ◽  
3T3 Fibroblasts ◽  
C Terminus

Although p21ras is localized to the plasma membrane, proteins it interacts with, such as the GTPase-activating proteins (GAPs) ras GAP and neurofibromin (NF1), are not, suggesting that one function of p21ras GTP may be to target such proteins to the plasma membrane. To investigate the effects of targeting ras GAP to the plasma membrane, ras C-terminal motifs sufficient for plasma membrane localization of p21ras were cloned onto the C terminus of ras GAP. Plasma membrane-targeted ras GAP is growth inhibitory to NIH 3T3 fibroblasts and COS cells. This growth inhibition correlates with GAP catalytic activity, since the plasma membrane-targeted C-terminal catalytic domain or the GAP-related domain of neurofibromin is inhibitory, whereas the similarly targeted N-terminal domain is not. Moreover, the inhibition is abrogated by the inactivating mutation L902I, which abolishes ras GAP catalytic activity. Coexpression of oncogenic mutant ras rescues cell viability, but the majority of rescued colonies are phenotypically untransformed. Furthermore, in focus assays, targeted ras GAP suppresses transformation by oncogenic mutant ras, and in reversion assays, targeted ras GAP can revert cells transformed by oncogenic mutant ras. Neither the targeted or nontargeted N-terminal domain nor the L902I mutant of ras GAP has any transforming activity. These data demonstrate that ras GAP can function as a negative regulator of ras and that plasma membrane localization potentiates this activity. However, if ras GAP is involved in the effector functions of p21ras, it can only be part of the effector complex for cell transformation.

scholarly journals Expression, purification, crystallization and preliminary crystallographic analysis of human myotubularin-related protein 3

2014 ◽  
Vol 70 (9) ◽  
pp. 1240-1243
Author(s):  
Ji Young Son ◽  
Jee Un Lee ◽  
Ki-Young Yoo ◽  
Woori Shin ◽  
Dong-Won Im ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Catalytic Domain ◽  
Family Members ◽  
Large Family ◽  
Crystallographic Analysis ◽  
Related Protein ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Related Proteins

Myotubularin-related proteins are a large family of phosphatases that have the catalytic activity of dephosphorylating the phospholipid molecules phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate. Each of the 14 family members contains a phosphatase catalytic domain, which is inactive in six family members owing to amino-acid changes in a key motif for the activity. All of the members also bear PH-GRAM domains, which have low homologies between them and have roles that are not yet clear. Here, the cloning, expression, purification and crystallization of human myotubularin-related protein 3 encompassing the PH-GRAM and the phosphatase catalytic domain are reported. Preliminary X-ray crystallographic analysis shows that the crystals diffracted to 3.30 Å resolution at a synchrotron X-ray source. The crystals belonged to space groupC2, with unit-cell parametersa= 323.3,b= 263.3,c= 149.4 Å, β = 109.7°.

scholarly journals Mutating His29, His125, His133 or His158 abolishes glycosylphosphatidylinositol-specific phospholipase D catalytic activity

2005 ◽  
Vol 391 (2) ◽  
pp. 285-289 ◽  
Author(s):  
Nandita S. Raikwar ◽  
Rosario F. Bowen ◽  
Mark A. Deeg
Keyword(s):  
Catalytic Activity ◽  
Phospholipase D ◽  
Catalytic Domain ◽  
Computer Modelling ◽  
Critical Role ◽  
Biochemical Properties ◽  
Catalytic Site ◽  
Wild Type ◽  
Histidine Residues ◽  
Hydrolysis Of

Glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) specifically cleaves GPIs. This phospholipase D is a secreted protein consisting of two domains: an N-terminal catalytic domain and a predicted C-terminal β-propeller. Although the biochemical properties of GPI-PLD have been extensively studied, its catalytic site has not been identified. We hypothesized that a histidine residue(s) may play a critical role in the catalytic activity of GPI-PLD, based on the observations that (i) Zn2+, which utilizes histidine residues for binding, is required for GPI-PLD catalytic activity, (ii) a phosphohistidine intermediate is involved in phospholipase D hydrolysis of phosphatidylcholine, (iii) computer modelling suggests a catalytic site containing histidine residues, and (iv) our observation that diethyl pyrocarbonate, which modifies histidine residues, inhibits GPI-PLD catalytic activity. Individual mutation of the ten histidine residues to asparagine in the catalytic domain of murine GPI-PLD resulted in three general phenotypes: not secreted or retained (His56 or His88), secreted with catalytic activity (His34, His81, His98 or His219) and secreted without catalytic activity (His29, His125, His133 or His158). Changing His133 but not His29, His125 or His158 to Cys resulted in a mutant that retained catalytic activity, suggesting that at least His133 is involved in Zn2+ binding. His133 and His158 also retained the biochemical properties of wild-type GPI-PLD including trypsin cleavage pattern and phosphorylation by protein kinase A. Hence, His29, His125, His133 and His158 are required for GPI-PLD catalytic activity.

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