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.

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.

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 d-Alanine–d-alanine ligase as a model for the activation of ATP-grasp enzymes by monovalent cations

2020 ◽  
Vol 295 (23) ◽  
pp. 7894-7904
Author(s):  
Jordan L. Pederick ◽  
Andrew P. Thompson ◽  
Stephen G. Bell ◽  
John B. Bruning
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Drug Target ◽  
Catalytic Mechanism ◽  
Thermus Thermophilus ◽  
Monovalent Cation ◽  
Antibiotic Drug ◽  
Domains Of Life ◽  
Alanine Dipeptide ◽  
Insight Into

The ATP-grasp superfamily of enzymes shares an atypical nucleotide-binding site known as the ATP-grasp fold. These enzymes are involved in many biological pathways in all domains of life. One ATP-grasp enzyme, d-alanine–d-alanine ligase (Ddl), catalyzes ATP-dependent formation of the d-alanyl–d-alanine dipeptide essential for bacterial cell wall biosynthesis and is therefore an important antibiotic drug target. Ddl is activated by the monovalent cation (MVC) K+, but despite its clinical relevance and decades of research, how this activation occurs has not been elucidated. We demonstrate here that activating MVCs bind adjacent to the active site of Ddl from Thermus thermophilus and used a combined biochemical and structural approach to characterize MVC activation. We found that TtDdl is a type II MVC-activated enzyme, retaining activity in the absence of MVCs. However, the efficiency of TtDdl increased ∼20-fold in the presence of activating MVCs, and it was maximally activated by K+ and Rb+ ions. A strict dependence on ionic radius of the MVC was observed, with Li+ and Na+ providing little to no TtDdl activation. To understand the mechanism of MVC activation, we solved crystal structures of TtDdl representing distinct catalytic stages in complex with K+, Rb+, or Cs+. Comparison of these structures with apo TtDdl revealed no evident conformational change on MVC binding. Of note, the identified MVC binding site is structurally conserved within the ATP-grasp superfamily. We propose that MVCs activate Ddl by altering the charge distribution of its active site. These findings provide insight into the catalytic mechanism of ATP-grasp enzymes.

scholarly journals The role of RelA (p65) threonine 505 phosphorylation in the regulation of cell growth, survival, and migration

2011 ◽  
Vol 22 (17) ◽  
pp. 3032-3040 ◽  
Author(s):  
Aichi Msaki ◽  
Ana M. Sánchez ◽  
Li Fang Koh ◽  
Benjamin Barré ◽  
Sonia Rocha ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Cellular Migration ◽  
Negative Regulator ◽  
Drug Induced ◽  
Conserved Residues ◽  
Cellular Processes ◽  
And Migration ◽  
Induced Apoptosis ◽  
Proliferation And Migration

The NF-κB family of transcription factors is a well-established regulator of the immune and inflammatory responses and also plays a key role in other cellular processes, including cell death, proliferation, and migration. Conserved residues in the trans-activation domain of RelA, which can be posttranslationally modified, regulate divergent NF-κB functions in response to different cellular stimuli. Using rela−/−mouse embryonic fibroblasts reconstituted with RelA, we find that mutation of the threonine 505 (T505) phospho site to alanine has wide-ranging effects on NF-κB function. These include previously described effects on chemotherapeutic drug-induced apoptosis, as well as new roles for this modification in autophagy, cell proliferation, and migration. This last effect was associated with alterations in the actin cytoskeleton and expression of cellular migration–associated genes such as WAVE3 and α-actinin 4. We also define a new component of cisplatin-induced, RelA T505–dependent apoptosis, involving induction of NOXA gene expression, an effect explained at least in part through induction of the p53 homologue, p73. Therefore, in contrast to other RelA phosphorylation events, which positively regulate NF-κB function, we identified RelA T505 phosphorylation as a negative regulator of its ability to induce diverse cellular processes such as apoptosis, autophagy, proliferation, and migration.

The Crystal Structure of the H48Q Active Site Mutant of Human Group IIA Secreted Phospholipase A2at 1.5 Å Resolution Provides an Insight into the Catalytic Mechanism†,‡

Biochemistry ◽  
2002 ◽  
Vol 41 (52) ◽  
pp. 15468-15476 ◽  
Author(s):  
Suzanne H. Edwards ◽  
Darren Thompson ◽  
Sharon F. Baker ◽  
Stephen P. Wood ◽  
David C. Wilton
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Human Group ◽  
Insight Into

The Role of Tyr248 Probed by Mutant Bovine Carboxypeptidase A:  Insight into the Catalytic Mechanism of Carboxypeptidase A†

Biochemistry ◽  
2001 ◽  
Vol 40 (34) ◽  
pp. 10197-10203 ◽  
Author(s):  
Jae Hyun Cho ◽  
Dong H. Kim ◽  
Do-Hyung Kim ◽  
Kyung Joo Lee ◽  
Kwan Yong Choi
Keyword(s):  
Catalytic Mechanism ◽  
Carboxypeptidase A ◽  
Insight Into

scholarly journals Structural Characterization of an S-enantioselective Imine Reductase from Mycobacterium Smegmatis

Biomolecules ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1130
Author(s):  
Timo Meyer ◽  
Nadine Zumbrägel ◽  
Christina Geerds ◽  
Harald Gröger ◽  
Hartmut H. Niemann
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Mycobacterium Smegmatis ◽  
Catalytic Mechanism ◽  
Building Blocks ◽  
Secondary Amines ◽  
Hydrophobic Amino Acids ◽  
High Degree

NADPH-dependent imine reductases (IREDs) are enzymes capable of enantioselectively reducing imines to chiral secondary amines, which represent important building blocks in the chemical and pharmaceutical industry. Since their discovery in 2011, many previously unknown IREDs have been identified, biochemically and structurally characterized and categorized into families. However, the catalytic mechanism and guiding principles for substrate specificity and stereoselectivity remain disputed. Herein, we describe the crystal structure of S-IRED-Ms from Mycobacterium smegmatis together with its cofactor NADPH. S-IRED-Ms belongs to the S-enantioselective superfamily 3 (SFam3) and is the first IRED from SFam3 to be structurally described. The data presented provide further evidence for the overall high degree of structural conservation between different IREDs of various superfamilies. We discuss the role of Asp170 in catalysis and the importance of hydrophobic amino acids in the active site for stereospecificity. Moreover, a separate entrance to the active site, potentially functioning according to a gatekeeping mechanism regulating access and, therefore, substrate specificity is described.

Sign in / Sign up

Export Citation Format

Share Document