Role of the potassium/lysine cationic center in catalysis and functional asymmetry in membrane-bound pyrophosphatases

2018 ◽  
Vol 475 (6) ◽  
pp. 1141-1158 ◽  
Author(s):  
Erika Artukka ◽  
Heidi H. Luoto ◽  
Alexander A. Baykov ◽  
Reijo Lahti ◽  
Anssi M. Malinen
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Systematic Analysis ◽  
Change Mechanism ◽  
Excess Substrate ◽  
Na Ions ◽  
Membrane Bound ◽  
A Site ◽  
Pyrophosphate Hydrolysis

Membrane-bound pyrophosphatases (mPPases), which couple pyrophosphate hydrolysis to transmembrane transport of H+ and/or Na+ ions, are divided into K+,Na+-independent, Na+-regulated, and K+-dependent families. The first two families include H+-transporting mPPases (H+-PPases), whereas the last family comprises one Na+-transporting, two Na+- and H+-transporting subfamilies (Na+-PPases and Na+,H+-PPases, respectively), and three H+-transporting subfamilies. Earlier studies of the few available model mPPases suggested that K+ binds to a site located adjacent to the pyrophosphate-binding site, but is substituted by the ε-amino group of an evolutionarily acquired lysine residue in the K+-independent mPPases. Here, we performed a systematic analysis of the K+/Lys cationic center across all mPPase subfamilies. An Ala → Lys replacement in K+-dependent mPPases abolished the K+ dependence of hydrolysis and transport activities and decreased these activities close to the level (4–7%) observed for wild-type enzymes in the absence of monovalent cations. In contrast, a Lys → Ala replacement in K+,Na+-independent mPPases conferred partial K+ dependence on the enzyme by unmasking an otherwise conserved K+-binding site. Na+ could partially replace K+ as an activator of K+-dependent mPPases and the Lys → Ala variants of K+,Na+-independent mPPases. Finally, we found that all mPPases were inhibited by excess substrate, suggesting strong negative co-operativity of active site functioning in these homodimeric enzymes; moreover, the K+/Lys center was identified as part of the mechanism underlying this effect. These findings suggest that the mPPase homodimer possesses an asymmetry of active site performance that may be an ancient prototype of the rotational binding-change mechanism of F-type ATPases.

Identification of SAS4 and SAS5, Two Genes That Regulate Silencing in Saccharomyces cerevisiae

Genetics ◽  
1999 ◽  
Vol 153 (1) ◽  
pp. 13-23 ◽  
Author(s):  
Eugenia Y Xu ◽  
Susan Kim ◽  
Kirstin Replogle ◽  
Jasper Rine ◽  
David H Rivier
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acute Myeloid Leukemia ◽  
Binding Site ◽  
Myeloid Leukemia ◽  
Null Alleles ◽  
Human Genes ◽  
Complementation Groups ◽  
A Site ◽  
Acute Myeloid

Abstract In Saccharomyces cerevisiae, chromatin-mediated silencing inactivates transcription of the genes at the HML and HMR cryptic mating-type loci and genes near telomeres. Mutations in the Rap1p and Abf1p binding sites of the HMR-E silencer (HMRa-e**) result in a loss of silencing at HMR. We characterized a collection of 15 mutations that restore the α-mating phenotype to MATα HMRa-e** strains. These mutations defined three complementation groups, two new groups and one group that corresponded to the previously identified SAS2 gene. We cloned the genes that complemented members of the new groups and identified two previously uncharacterized genes, which we named SAS4 and SAS5. Neither SAS4 nor SAS5 was required for viability. Null alleles of SAS4 and SAS5 restored SIR4-dependent silencing at HMR, establishing that each is a regulator of silencing. Null alleles of SAS4 and SAS5 bypassed the role of the Abf1p binding site of the HMR-E silencer but not the role of the ACS or Rap1p binding site. Previous analysis indicated that SAS2 is homologous to a human gene that is a site of recurring translocations involved in acute myeloid leukemia. Similarly, SAS5 is a member of a gene family that included two human genes that are the sites of recurring translocations involved in acute myeloid leukemia.

scholarly journals Substrate-modulated Cytochrome P450 17A1 and Cytochrome b5 Interactions Revealed by NMR

2013 ◽  
Vol 288 (23) ◽  
pp. 17008-17018 ◽  
Author(s):  
D. Fernando Estrada ◽  
Jennifer S. Laurence ◽  
Emily E. Scott
Keyword(s):  
Cytochrome P450 ◽  
Binding Site ◽  
Active Site ◽  
Catalytic Domain ◽  
Cytochrome B5 ◽  
Structural Basis ◽  
Reaction Conditions ◽  
Reversible Binding ◽  
Important Interaction

The membrane heme protein cytochrome b5 (b5) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Here the interactions between the soluble domain of microsomal b5 and the catalytic domain of the bifunctional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b5, and an androgen-forming lyase reaction that is facilitated 10-fold by b5. NMR chemical shift mapping of b5 titrations with CYP17A1 indicates that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b5 residues (Glu-48 or Glu-49) or the corresponding cationic CYP17A1 residues (Arg-347, Arg-358, or Arg-449). Cytochrome b5 binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase. To probe the differential effects of b5 on the two CYP17A1-mediated reactions and, thus, communication between the superficial b5 binding site and the buried CYP17A1 active site, CYP17A1/b5 complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17α-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b5 binding site.

Role of Arginine Residues in the Active Site of the Membrane-Bound Lytic Transglycosylase B fromPseudomonas aeruginosa†

Biochemistry ◽  
2006 ◽  
Vol 45 (7) ◽  
pp. 2129-2138 ◽  
Author(s):  
Christopher W. Reid ◽  
Neil T. Blackburn ◽  
Anthony J. Clarke
Keyword(s):  
Active Site ◽  
Lytic Transglycosylase ◽  
Arginine Residues ◽  
Membrane Bound

scholarly journals Structural basis of adenylyl cyclase 9 activation

2021 ◽  
Author(s):  
Chao Qi ◽  
Pia Lavriha ◽  
Ved Mehta ◽  
Basavraj Khanppnavar ◽  
Inayathulla Mohammed ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Binding Site ◽  
Adenylyl Cyclase ◽  
Conformational Changes ◽  
Structural Basis ◽  
Structural Studies ◽  
Nucleotide Analogue ◽  
C Terminus ◽  
Membrane Bound ◽  
A Site

Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gαs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPαS, (iv) bound to Gαs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gαs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four new conformations show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation.

scholarly journals Mapping the Substrate Binding Site of Phenylacetone Monooxygenase from Thermobifida fusca by Mutational Analysis

2011 ◽  
Vol 77 (16) ◽  
pp. 5730-5738 ◽  
Author(s):  
Hanna M. Dudek ◽  
Gonzalo de Gonzalo ◽  
Daniel E. Torres Pazmiño ◽  
Piotr Stępniak ◽  
Lucjan S. Wyrwicz ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Binding Site ◽  
Active Site ◽  
Structural Model ◽  
Mutational Analysis ◽  
Substrate Binding ◽  
Thermobifida Fusca ◽  
Content Type ◽  
Substrate Binding Site

ABSTRACTBaeyer-Villiger monooxygenases catalyze oxidations that are of interest for biocatalytic applications. Among these enzymes, phenylacetone monooxygenase (PAMO) fromThermobifida fuscais the only protein showing remarkable stability. While related enzymes often present a broad substrate scope, PAMO accepts only a limited number of substrates. Due to the absence of a substrate in the elucidated crystal structure of PAMO, the substrate binding site of this protein has not yet been defined. In this study, a structural model of cyclopentanone monooxygenase, which acts on a broad range of compounds, has been prepared and compared with the structure of PAMO. This revealed 15 amino acid positions in the active site of PAMO that may account for its relatively narrow substrate specificity. We designed and analyzed 30 single and multiple mutants in order to verify the role of these positions. Extensive substrate screening revealed several mutants that displayed increased activity and altered regio- or enantioselectivity in Baeyer-Villiger reactions and sulfoxidations. Further substrate profiling resulted in the identification of mutants with improved catalytic properties toward synthetically attractive compounds. Moreover, the thermostability of the mutants was not compromised in comparison to that of the wild-type enzyme. Our data demonstrate that the positions identified within the active site of PAMO, namely, V54, I67, Q152, and A435, contribute to the substrate specificity of this enzyme. These findings will aid in more dedicated and effective redesign of PAMO and related monooxygenases toward an expanded substrate scope.

scholarly journals Role of Serine 286 in cosubstrate binding and catalysis of a flavonol O-methyltransferase

2004 ◽  
Vol 82 (5) ◽  
pp. 531-537 ◽  
Author(s):  
Jack Kornblatt ◽  
Ingrid Muzac ◽  
Yoongho Lim ◽  
Joong Hoon Ahn ◽  
Ragai K Ibrahim
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Photoaffinity Labeling ◽  
Single Amino Acid ◽  
Hydroxyl Groups ◽  
Cdna Clones ◽  
Subunit Interface ◽  
Single Amino Acid Residue ◽  
Chrysosplenium Americanum

O-Methyltransferases catalyze the transfer of the methyl groups of S-adenosyl-L-methionine to specific hydroxyl groups of several classes of flavonoid compounds. Of the several cDNA clones isolated from a Chrysosplenium americanum library, FOMT3′ encodes the 3′/5′-O-methylation of partially methylated flavonols. The recombinant protein of another clone, FOMTx which differs from FOMT3′ by a single amino acid residue (Ser286Arg) exhibits no enzymatic activity towards any of the flavonoid substrates tested. Replacement of Ser 286 in FOMT3′ with either Ala, Leu, Lys or Thr, almost abolished O-methyltransferase activity. In contrast with FOMT3′, no photoaffinity labeling could be achieved using [14CH3]AdoMet with the mutant recombinant proteins indicating that Ser 286 is also required for cosubstrate binding. These results are corroborated by isothermal titration microcalorimetry measurements. Circular dichroism spectra ruled out any significant conformational differences in the secondary structures of both FOMT3′ and Ser286Arg. Modeling FOMT3′ on the structure of chalcone methyltransferase indicates that serine 286 is greater than 10 Å from any of the residues of the active site or the AdoMet binding site of FOMT3′. At the same time, residues 282 to 290 are conserved in most of the Chrysosplenium americanum OMTs. These residues form a large part of the subunit interface, and at least five of these residues are within 4 Å of the opposing subunit. It would appear, therefore, that mutations in Ser286 exert their influence by altering the contacts between the subunits and that these contacts are necessary for maintaining the integrety of the AdoMet binding site and active site of this group of enzymes. Key words: flavonoids, O-methyltransferase, photoaffinity labeling.

scholarly journals Destruction, Reconstruction and Resistance: The Skin and the Protean Body in Pedro Almodóvar’s Body Horror The Skin I Live In

Humanities ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 54
Author(s):  
Subarna Mondal
Keyword(s):  
Active Site ◽  
The Body ◽  
The Other ◽  
Human Anatomy ◽  
Surgical Operation ◽  
Mise En Scene ◽  
History Of ◽  
Female Bodies ◽  
A Site

The instinct to tame and preserve and the longing for eternal beauty makes skin a crucial element in the genre of the Body Horror. By applying a gendered reading to the art of destruction and reconstruction of an ephemeral body, this paper explores the significant role of skin that clothes a protean body in Almodóvar’s unconventional Body Horror, “The Skin I Live In” (2011). Helpless vulnerable female bodies stretched on beds and close shots of naked perfect skin of those bodies are a frequent feature in Almodóvar films. Skin stained and blotched in “Tie Me Up! Tie Me Down!” (1989), nurtured and replenished in “Talk to Her” (2002), patched up and stitched in “The Skin I Live In”, becomes a key ingredient in Almodóvar’s films that celebrate the fluidity of human anatomy and sexuality. The article situates “The Skin I Live In” in the filmic continuum of Body Horrors that focus primarily on skin, beginning with Alfred Hitchcock’s “Psycho” (1960), and touching on films like Jonathan Demme’s “The Silence of the Lambs” (1991) and Tom Tykwer’s “Perfume: The Story of a Murderer” (2006) and attempts to understand how the exploited bodies that have been culturally and socially subjugated have shaped the course of the history of Body Horrors in cinema. In “The Skin I Live In” the destruction of Vicente’s body and its recreation into Vera follow a mad scientist’s urge to dominate an unattainable body, but this ghastly assault on the body has the onscreen appearance of a routine surgical operation by an expert cosmetologist in a well-lit, sanitized mise-en-scène, suggesting that the uncanny does not need a dungeon to lurk in. The exploited body on the other hand may be seen not as a passive victim, but as a site of alterity and rebellion. Anatomically a complete opposite of Frankenstein’s Creature, Vicente/Vera’s body, perfect, beautiful but beset with a problematized identity, is etched with the history of conversion, suppression, and the eternal quest for an ephemeral object. Yet it also acts as an active site of resistance.

Evidence for the role of fibronectin in amphibian gastrulation

Development ◽  
1985 ◽  
Vol 89 (Supplement) ◽  
pp. 211-227
Author(s):  
J. C. Boucaut ◽  
T. Darribere ◽  
Shi De Li ◽  
H. Boulekbache ◽  
K. M. Yamada ◽  
...  
Keyword(s):  
Cell Migration ◽  
Binding Site ◽  
Synthetic Peptide ◽  
Complete Inhibition ◽  
Cell Binding ◽  
Mesodermal Cell ◽  
Local Inversion ◽  
A Site ◽  
Fibrillar Network

In amphibian embryos, fibronectin (FN) assembles as a fibrillar network on the roof of the blastocoel cavity, preceding mesodermal cell migration. Local inversion of the ectoderm to produce a site where no FN is available prevents mesodermal cell migration. Microinjection of monovalent antibodies to FN arrests gastrulation. A complete inhibition of mesodermal cell migration is obtained after microinjection of a synthetic peptide containing the cell binding site sequence of FN. Prevention of interactions between receptors and FN appears to be the primary cause for blockage of gastrulation.

scholarly journals Crystal structure of human phosphoribosylpyrophosphate synthetase 1 reveals a novel allosteric site

2006 ◽  
Vol 401 (1) ◽  
pp. 39-47 ◽  
Author(s):  
Sheng Li ◽  
Yongcheng Lu ◽  
Baozhen Peng ◽  
Jianping Ding
Keyword(s):  
Enzymatic Activity ◽  
Binding Site ◽  
Active Site ◽  
Atp Binding ◽  
Allosteric Site ◽  
Nucleotide Synthesis ◽  
Structural Stabilization ◽  
Atp Binding Site ◽  
Phosphate Ions ◽  
A Site

PRPP (phosphoribosylpyrophosphate) is an important metabolite essential for nucleotide synthesis and PRS (PRPP synthetase) catalyses synthesis of PRPP from R5P (ribose 5-phosphate) and ATP. The enzymatic activity of PRS is regulated by phosphate ions, divalent metal cations and ADP. In the present study we report the crystal structures of recombinant human PRS1 in complexes with SO42− ions alone and with ATP, Cd2+ and SO42− ions respectively. The AMP moiety of ATP binds at the ATP-binding site, and a Cd2+ ion binds at the active site and in a position to interact with the β- and γ-phosphates of ATP. A SO42− ion, an analogue of the activator phosphate, was found to bind at both the R5P-binding site and the allosteric site defined previously. In addi-tion, an extra SO42− binds at a site at the dimer interface between the ATP-binding site and the allosteric site. Binding of this SO42− stabilizes the conformation of the flexible loop at the active site, leading to the formation of the active, open conformation which is essential for binding of ATP and initiation of the catalytic reaction. This is the first time that structural stabilization at the active site caused by binding of an activator has been observed. Structural and biochemical data show that mutations of some residues at this site influence the binding of SO42− and affect the enzymatic activity. The results in the present paper suggest that this new SO42−-binding site is a second allosteric site to regulate the enzymatic activity which might also exist in other eukaryotic PRSs (except plant PRSs of class II), but not in bacterial PRSs.

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