Conserved residues of liquefying α-amylases are concentrated in the vicinity of active site

1990 ◽  
Vol 166 (1) ◽  
pp. 61-65 ◽  
Author(s):  
Mauno Vihinen ◽  
Pekka Mäntsälä
Keyword(s):  
Active Site ◽  
Conserved Residues

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 GlcNAcstatins are nanomolar inhibitors of human O-GlcNAcase inducing cellular hyper-O-GlcNAcylation

2009 ◽  
Vol 420 (2) ◽  
pp. 221-227 ◽  
Author(s):  
Helge C. Dorfmueller ◽  
Vladimir S. Borodkin ◽  
Marianne Schimpl ◽  
Daan M. F. van Aalten
Keyword(s):  
Active Site ◽  
Cell Biology ◽  
Rational Design ◽  
Catalytic Mechanism ◽  
Conserved Residues ◽  
Cellular Processes ◽  
Acetyl Group ◽  
Nanomolar Range ◽  
Insight Into

O-GlcNAcylation is an essential, dynamic and inducible post-translational glycosylation of cytosolic proteins in metazoa and can show interplay with protein phosphorylation. Inhibition of OGA (O-GlcNAcase), the enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, is a useful strategy to probe the role of this modification in a range of cellular processes. In the present study, we report the rational design and evaluation of GlcNAcstatins, a family of potent, competitive and selective inhibitors of human OGA. Kinetic experiments with recombinant human OGA reveal that the GlcNAcstatins are the most potent human OGA inhibitors reported to date, inhibiting the enzyme in the sub-nanomolar to nanomolar range. Modification of the GlcNAcstatin N-acetyl group leads to up to 160-fold selectivity against the human lysosomal hexosaminidases which employ a similar substrate-assisted catalytic mechanism. Mutagenesis studies in a bacterial OGA, guided by the structure of a GlcNAcstatin complex, provides insight into the role of conserved residues in the human OGA active site. GlcNAcstatins are cell-permeant and, at low nanomolar concentrations, effectively modulate intracellular O-GlcNAc levels through inhibition of OGA, in a range of human cell lines. Thus these compounds are potent selective tools to study the cell biology of O-GlcNAc.

scholarly journals The interplay of DNA binding, ATP hydrolysis and helicase activities of the archaeal MCM helicase

2011 ◽  
Vol 436 (2) ◽  
pp. 409-414 ◽  
Author(s):  
Li Phing Liew ◽  
Stephen D. Bell
Keyword(s):  
Dna Binding ◽  
Active Site ◽  
Atp Hydrolysis ◽  
Sulfolobus Solfataricus ◽  
Active Form ◽  
Dna Helicase ◽  
Atpase Domain ◽  
Minichromosome Maintenance ◽  
Conserved Residues ◽  
Mcm Helicase

The MCM (minichromosome maintenance) proteins of archaea are widely believed to be the replicative DNA helicase of these organisms. Most archaea possess a single MCM orthologue that forms homo-multimeric assemblies with a single hexamer believed to be the active form. In the present study we characterize the roles of highly conserved residues in the ATPase domain of the MCM of the hyperthermophilic archaeon Sulfolobus solfataricus. Our results identify a potential conduit for communicating DNA-binding information to the ATPase active site.

scholarly journals Structure of replicating SARS-CoV-2 polymerase

2020 ◽  
Author(s):  
Hauke S. Hillen ◽  
Goran Kokic ◽  
Lucas Farnung ◽  
Christian Dienemann ◽  
Dimitry Tegunov ◽  
...  
Keyword(s):  
Active Site ◽  
Viral Proteins ◽  
Microscopic Structure ◽  
Rdrp Activity ◽  
Electron Microscopic ◽  
Rna Dependent Rna Polymerase ◽  
Conserved Residues ◽  
Inhibitory Mechanisms ◽  
Active Site Cleft ◽  
Positively Charged

The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. Here we present the cryo-electron microscopic structure of the SARS-CoV-2 RdRp in its replicating form. The structure comprises the viral proteins nsp12, nsp8, and nsp7, and over two turns of RNA template-product duplex. The active site cleft of nsp12 binds the first turn of RNA and mediates RdRp activity with conserved residues. Two copies of nsp8 bind to opposite sides of the cleft and position the RNA duplex as it exits. Long helical extensions in nsp8 protrude along exiting RNA, forming positively charged ‘sliding poles’ that may enable processive replication of the long coronavirus genome. Our results will allow for a detailed analysis of the inhibitory mechanisms used by antivirals such as remdesivir, which is currently in clinical trials for the treatment of coronavirus disease 2019 (COVID-19).

scholarly journals Serendipitous crystallization and structure determination of cyanase (CynS) fromSerratia proteamaculans

2015 ◽  
Vol 71 (4) ◽  
pp. 471-476 ◽  
Author(s):  
Agata Butryn ◽  
Gabriele Stoehr ◽  
Christian Linke-Winnebeck ◽  
Karl-Peter Hopfner
Keyword(s):  
Escherichia Coli ◽  
Carbon Dioxide ◽  
Structure Determination ◽  
Active Site ◽  
Mass Spectrometric ◽  
Mass Spectrometric Analysis ◽  
Spectrometric Analysis ◽  
Conserved Residues ◽  
Serratia Proteamaculans

Cyanate hydratase (CynS) catalyzes the decomposition of cyanate and bicarbonate into ammonia and carbon dioxide. Here, the serendipitous crystallization of CynS fromSerratia proteamaculans(SpCynS) is reported. SpCynS was crystallized as an impurity and its identity was determined using mass-spectrometric analysis. The crystals belonged to space groupP1 and diffracted to 2.1 Å resolution. The overall structure of SpCynS is very similar to a previously determined structure of CynS fromEscherichia coli. Density for a ligand bound to the SpCynS active site was observed, but could not be unambiguously identified. Additionally, glycerol molecules bound at the entry to the active site of the enzyme indicate conserved residues that might be important for the trafficking of substrates and products.

scholarly journals Substitution of Conserved Residues within the Active Site Alters the Cleavage Religation Equilibrium of DNA Topoisomerase I

2004 ◽  
Vol 279 (52) ◽  
pp. 54069-54078 ◽  
Author(s):  
William C. Colley ◽  
Marie van der Merwe ◽  
John R. Vance ◽  
Alex B. Burgin ◽  
Mary-Ann Bjornsti
Keyword(s):  
Active Site ◽  
Topoisomerase I ◽  
Dna Topoisomerase I ◽  
Dna Topoisomerase ◽  
Conserved Residues

Structure and mechanism of the chalcogen-detoxifying protein TehB from Escherichia coli

2011 ◽  
Vol 435 (1) ◽  
pp. 85-91 ◽  
Author(s):  
Hassanul G. Choudhury ◽  
Alexander D. Cameron ◽  
So Iwata ◽  
Konstantinos Beis
Keyword(s):  
Escherichia Coli ◽  
Reaction Mechanism ◽  
Active Site ◽  
Kinetic Data ◽  
Protein Analysis ◽  
Nucleophilic Attack ◽  
Conserved Residues ◽  
Low Levels ◽  
Living Organisms ◽  
Derivatives Of

The oxyanion derivatives of the chalcogens tellurium and selenium are toxic to living organisms even at very low levels. Bacteria have developed mechanisms to overcome their toxicity by methylating them. The structure of TehB from Escherichia coli has been determined in the presence of the cofactor analogues SAH (S-adenosylhomocysteine) and sinefungin (a non-hydrolysable form of S-adenosyl-L-methionine) at 1.48 Å (1 Å=0.1 nm) and 1.9 Å respectively. Interestingly, our kinetic data show that TehB does not discriminate between selenium or tellurite oxyanions, making it a very powerful detoxifying protein. Analysis of the active site has identified three conserved residues that are capable of binding and orientating the metals for nucleophilic attack: His176, Arg177 and Arg184. Mutagenesis studies revealed that the H176A and R184A mutants retained most of their activity, whereas the R177A mutant had 65% of its activity abolished. Based on the structure and kinetic data we propose an SN2 nucleophilic attack reaction mechanism. These data provide the first molecular understanding of the detoxification of chalcogens by bacteria.

Sign in / Sign up

Export Citation Format

Share Document