Geometric and Electronic Structure Studies of the Binuclear Nonheme Ferrous Active Site of Toluene-4-monooxygenase: Parallels with Methane Monooxygenase and Insight into the Role of the Effector Proteins in O2Activation

2008 ◽  
Vol 130 (22) ◽  
pp. 7098-7109 ◽  
Author(s):  
Jennifer K. Schwartz ◽  
Pin-pin Wei ◽  
Kevin H. Mitchell ◽  
Brian G. Fox ◽  
Edward I. Solomon
Keyword(s):  
Electronic Structure ◽  
Active Site ◽  
Methane Monooxygenase ◽  
Effector Proteins ◽  
Insight Into

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 1H NMR Spectroscopy of [FeFe] Hydrogenase: Insight into the Electronic Structure of the Active Site

2017 ◽  
Vol 140 (1) ◽  
pp. 131-134 ◽  
Author(s):  
Sigrun Rumpel ◽  
Enrico Ravera ◽  
Constanze Sommer ◽  
Edward Reijerse ◽  
Christophe Farès ◽  
...  
Keyword(s):  
Electronic Structure ◽  
Nmr Spectroscopy ◽  
1H Nmr ◽  
Active Site ◽  
1H Nmr Spectroscopy ◽  
Insight Into

scholarly journals A viral ring nuclease anti-CRISPR subverts type III CRISPR immunity

2019 ◽  
Author(s):  
Januka S Athukoralage ◽  
Stephen McMahon ◽  
Changyi Zhang ◽  
Sabine Grüschow ◽  
Shirley Graham ◽  
...  
Keyword(s):  
Active Site ◽  
Cyclic Nucleotides ◽  
Effector Proteins ◽  
Broad Host Range ◽  
Type Iii ◽  
Signalling Molecules ◽  
New Family ◽  
Signalling Molecule ◽  
Defence Enzymes

ABSTRACTThe CRISPR system provides adaptive immunity against mobile genetic elements in bacteria and archaea. On detection of viral RNA, type III CRISPR systems generate a cyclic oligoadenylate (cOA) second messenger1–3, activating defence enzymes and sculpting a powerful antiviral response that can drive viruses to extinction4,5. Cyclic nucleotides are increasingly implicated as playing an important role in host-pathogen interactions6,7. Here, we identify a widespread new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease (AcrIII-1) is the first Acr described for type III CRISPR systems and is widely distributed in archaeal and bacterial viruses, and proviruses. The enzyme uses a novel fold to bind cA4specifically and utilizes a conserved active site to rapidly cleave the signalling molecule, allowing viruses to neutralise the type III CRISPR defence system. The AcrIII-1 family has a broad host range as it targets cA4signalling molecules rather than specific CRISPR effector proteins. This study highlights the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.

Reaction of threonine synthase with the substrate analogue 2-amino-5-phosphonopentanoate: implications into the proton transfer at the active site

2019 ◽  
Vol 167 (4) ◽  
pp. 357-364
Author(s):  
Yasuhiro Machida ◽  
Takeshi Murakawa ◽  
Akiko Sakai ◽  
Mitsuo Shoji ◽  
Yasuteru Shigeta ◽  
...  
Keyword(s):  
Catalytic Reaction ◽  
Active Site ◽  
Catalytic Mechanism ◽  
The Other ◽  
Amino Group ◽  
Threonine Synthase ◽  
Spectral Changes ◽  
Proton Migration ◽  
Insight Into

Abstract Threonine synthase catalyses the conversion of O-phospho-l-homoserine and a water molecule to l-threonine and has the most complex catalytic mechanism among the pyridoxal 5′-phosphate-dependent enzymes. In order to study the less-characterized earlier stage of the catalytic reaction, we studied the reaction of threonine synthase with 2-amino-5-phosphonopentanoate, which stops the catalytic reaction at the enamine intermediate. The global kinetic analysis of the triphasic spectral changes showed that, in addition to the theoretically expected pathway, the carbanion is rapidly reprotonated at Cα to form an aldimine distinct from the external aldimine directly formed from the Michaelis complex. The Kd for the binding of inhibitor to the enzyme decreased with increasing pH, showing that the 2-amino-group-unprotonated form of the ligand binds to the enzyme. On the other hand, the rate constants for the proton migration steps within the active site are independent of the solvent pH, indicating that protons are shared by the active dissociative groups and are not exchanged with the solvent during the course of catalysis. This gives an insight into the role of the phosphate group of the substrate, which may increase the basicity of the ε-amino group of the catalytic lysine residue in the active site.

Spectroscopic and electronic structure studies of the role of active site interactions in the decarboxylation reaction of α-keto acid-dependent dioxygenases

2006 ◽  
Vol 100 (12) ◽  
pp. 2108-2116 ◽  
Author(s):  
Michael L. Neidig ◽  
Christina D. Brown ◽  
Michael Kavana ◽  
Oliver W. Choroba ◽  
Jonathan B. Spencer ◽  
...  
Keyword(s):  
Electronic Structure ◽  
Active Site ◽  
Keto Acid ◽  
Decarboxylation Reaction

Spectroscopic and Density Functional Studies of the Red Copper Site in Nitrosocyanin:  Role of the Protein in Determining Active Site Geometric and Electronic Structure

2005 ◽  
Vol 127 (10) ◽  
pp. 3531-3544 ◽  
Author(s):  
Lipika Basumallick ◽  
Ritimukta Sarangi ◽  
Serena DeBeer George ◽  
Brad Elmore ◽  
Alan B. Hooper ◽  
...  
Keyword(s):  
Electronic Structure ◽  
Active Site ◽  
Density Functional ◽  
Functional Studies ◽  
Copper Site
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