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 Catalytic and structural contributions for glutathione-binding residues in a Delta class glutathione S-transferase

2004 ◽  
Vol 382 (2) ◽  
pp. 751-757 ◽  
Author(s):  
Pakorn WINAYANUWATTIKUN ◽  
Albert J. KETTERMAN
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Structural Integrity ◽  
Catalytic Mechanism ◽  
Tertiary Structure ◽  
Site Directed Mutagenesis ◽  
Active Site Residues ◽  
Anopheles Dirus ◽  
Steady State Kinetics ◽  
Cellular Detoxification

Glutathione S-transferases (GSTs) are dimeric proteins that play a major role in cellular detoxification. The GSTs in mosquito Anopheles dirus species B, an important malaria vector in South East Asia, are of interest because they can play an important role in insecticide resistance. In the present study, we characterized the Anopheles dirus (Ad)GST D3-3 which is an alternatively spliced product of the adgst1AS1 gene. The data from the crystal structure of GST D3-3 shows that Ile-52, Glu-64, Ser-65, Arg-66 and Met-101 interact directly with glutathione. To study the active-site function of these residues, alanine substitution site-directed mutagenesis was performed resulting in five mutants: I52A (Ile-52→Ala), E64A, S65A, R66A and M101A. Interestingly, the E64A mutant was expressed in Escherichia coli in inclusion bodies, suggesting that this residue is involved with the tertiary structure or folding property of this enzyme. However, the I52A, S65A, R66A and M101A mutants were purified by glutathione affinity chromatography and the enzyme activity characterized. On the basis of steady-state kinetics, difference spectroscopy, unfolding and refolding studies, it was concluded that these residues: (1) contribute to the affinity of the GSH-binding site (‘G-site’) for GSH, (2) influence GSH thiol ionization, (3) participate in kcat regulation by affecting the rate-limiting step of the reaction, and in the case of Ile-52 and Arg-66, influenced structural integrity and/or folding of the enzyme. The structural perturbations from these mutants are probably transmitted to the hydrophobic-substrate-binding site (‘H-site’) through changes in active site topology or through effects on GSH orientation. Therefore these active site residues appear to contribute to various steps in the catalytic mechanism, as well as having an influence on the packing of the protein.

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.

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

scholarly journals Crystal structure of the RNA 2′,3′-cyclic phosphodiesterase fromDeinococcus radiodurans

2017 ◽  
Vol 73 (5) ◽  
pp. 276-280 ◽  
Author(s):  
Wanchun Han ◽  
Jiahui Cheng ◽  
Congli Zhou ◽  
Yuejin Hua ◽  
Ye Zhao
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Deinococcus Radiodurans ◽  
Sulfate Ion ◽  
Open Conformation ◽  
Domains Of Life

2′,3′-Cyclic phosphodiesterase (CPDase) homologues have been found in all domains of life and are involved in diverse RNA and nucleotide metabolisms. The CPDase fromDeinococcus radioduranswas crystallized and the crystals diffracted to 1.6 Å resolution, which is the highest resolution currently known for a CPDase structure. Structural comparisons revealed that the enzyme is in an open conformation in the absence of substrate. Nevertheless, the active site is well formed, and the representative motifs interact with sulfate ion, which suggests a conserved catalytic mechanism.

scholarly journals Active-site MMP-selective antibody inhibitors discovered from convex paratope synthetic libraries

2016 ◽  
Vol 113 (52) ◽  
pp. 14970-14975 ◽  
Author(s):  
Dong Hyun Nam ◽  
Carlos Rodriguez ◽  
Albert G. Remacle ◽  
Alex Y. Strongin ◽  
Xin Ge
Keyword(s):  
Active Site ◽  
Drug Target ◽  
Catalytic Mechanism ◽  
Catalytic Domain ◽  
Selective Inhibitors ◽  
Protease Cleavage ◽  
Pharmacological Targets ◽  
Antibody Libraries ◽  
Inhibitory Antibodies ◽  
Complementarity Determining Region

Proteases are frequent pharmacological targets, and their inhibitors are valuable drugs in multiple pathologies. The catalytic mechanism and the active-site fold, however, are largely conserved among the protease classes, making the development of the selective inhibitors exceedingly challenging. In our departure from the conventional strategies, we reviewed the structure of known camelid inhibitory antibodies, which block enzyme activities via their unusually long, convex-shaped paratopes. We synthesized the human Fab antibody library (over 1.25 × 109 individual variants) that carried the extended, 23- to 27-residue, complementarity-determining region (CDR)–H3 segments. As a proof of principle, we used the catalytic domain of matrix metalloproteinase-14 (MMP-14), a promalignant protease and a drug target in cancer, as bait. In our screens, we identified 20 binders, of which 14 performed as potent and selective inhibitors of MMP-14 rather than as broad-specificity antagonists. Specifically, Fab 3A2 bound to MMP-14 in the vicinity of the active pocket with a high 4.8 nM affinity and was similarly efficient (9.7 nM) in inhibiting the protease cleavage activity. We suggest that the convex paratope antibody libraries described here could be readily generalized to facilitate the design of the antibody inhibitors to many additional enzymes.

A QM/MM Investigation of the Catalytic Mechanism of Acetylene Hydratase: Insights into Engineering a More Effective Enzyme

2020 ◽  
Author(s):  
wenyuan zhang ◽  
Eric A.C. Bushnell
Keyword(s):  
Active Site ◽  
Cluster Model ◽  
Reaction Rate ◽  
Catalytic Mechanism ◽  
Single Step ◽  
Wild Type ◽  
Step Process ◽  
Rate Determining Step ◽  
Binding Phase ◽  
Insight Into

In the present investigation, a QM/MM approach was used to better understand the effect of the second environmental shell of the active site on the catalytic conversion of acetylene to acetaldehyde by acetylene hydratase (AH). In addition, the effect of substituting W-coordinating sulfur atoms with selenium atoms was done to provide insight into the influence of the W-coordinating atoms on the catalytic reaction. From the results, it found that the presence of the second shell environment had a significant effect on the reaction. Specifically, in the absence of the MM second shell environment(i.e., QM-cluster model), the reaction rate-determining step is defined by the first proton transfer step. In contrast, for the QM/MM model, the rate-determining step is defined by the water attacking step. Moreover, with the presence of the MM second shell environment, a key intermediate found in the DFT-cluster investigation does not exist in the QM/MM investigation. Rather, what was a two-step process in the DFT-cluster study was calculated to occur in a single step for the QM/MM study. Regarding the sulfur to selenium substitutions, it was found that Gibbs energy for the acetylene binding phase was significantly affected. Notably, the trans-position selenium made the binding of acetylene 65.6 kJ mol-1 less endergonic. Moreover, the overall reaction became 38.2 kJ mol-1 less endergonic compared to the wild type (WT) AH model. Thus, the substitution of key W-coordinating sulfur atoms with selenium atoms may offer a means to enhance the catalytic mechanism of AH considerably.

scholarly journals Crystal Structure ofN-Succinylarginine Dihydrolase AstB, Bound to Substrate and Product, an Enzyme from the Arginine Catabolic Pathway ofEscherichia coli

2005 ◽  
Vol 280 (16) ◽  
pp. 15800-15808 ◽  
Author(s):  
Ante Tocilj ◽  
Joseph D. Schrag ◽  
Yunge Li ◽  
Barbara L. Schneider ◽  
Larry Reitzer ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Catabolic Pathway ◽  
Major Pathway ◽  
Product Release ◽  
Flexible Loop ◽  
Ammonia Release ◽  
Arginine Catabolism ◽  
Insight Into

The ammonia-producing arginine succinyltransferase pathway is the major pathway inEscherichia coliand related bacteria for arginine catabolism as a sole nitrogen source. This pathway consists of five steps, each catalyzed by a distinct enzyme. Here we report the crystal structure ofN-succinylarginine dihydrolase AstB, the second enzyme of the arginine succinyltransferase pathway, providing the first structural insight into enzymes from this pathway. The enzyme exhibits a pseudo 5-fold symmetric α/β propeller fold of circularly arranged ββαβ modules enclosing the active site. The crystal structure indicates clearly that this enzyme belongs to the amidinotransferase (AT) superfamily and that the active site contains a Cys–His-Glu triad characteristic of the AT superfamily. Structures of the complexes of AstB with the reaction product and a C365S mutant with bound theN-succinylarginine substrate suggest a catalytic mechanism that consists of two cycles of hydrolysis and ammonia release, with each cycle utilizing a mechanism similar to that proposed for arginine deiminases. Like other members of the AT superfamily of enzymes, AstB possesses a flexible loop that is disordered in the absence of substrate and assumes an ordered conformation upon substrate binding, shielding the ligand from the bulk solvent, thereby controlling substrate access and product release.

scholarly journals Structures of the hydrolase domain of zebrafish 10-formyltetrahydrofolate dehydrogenase and its complexes reveal a complete set of key residues for hydrolysis and product inhibition

2015 ◽  
Vol 71 (4) ◽  
pp. 1006-1021 ◽  
Author(s):  
Chien-Chih Lin ◽  
Phimonphan Chuankhayan ◽  
Wen-Ni Chang ◽  
Tseng-Ting Kao ◽  
Hong-Hsiang Guan ◽  
...  
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Product Inhibition ◽  
Product Specificity ◽  
Product Release ◽  
Terminal Domain ◽  
Complete Set ◽  
Formyltetrahydrofolate Dehydrogenase ◽  
Key Residues

10-Formyltetrahydrofolate dehydrogenase (FDH), which is composed of a small N-terminal domain (Nt-FDH) and a large C-terminal domain, is an abundant folate enzyme in the liver and converts 10-formyltetrahydrofolate (10-FTHF) to tetrahydrofolate (THF) and CO2. Nt-FDH alone possesses a hydrolase activity, which converts 10-FTHF to THF and formate in the presence of β-mercaptoethanol. To elucidate the catalytic mechanism of Nt-FDH, crystal structures of apo-form zNt-FDH from zebrafish and its complexes with the substrate analogue 10-formyl-5,8-dideazafolate (10-FDDF) and with the products THF and formate have been determined. The structures reveal that the conformations of three loops (residues 86–90, 135–143 and 200–203) are altered upon ligand (10-FDDF or THF) binding in the active site. The orientations and geometries of key residues, including Phe89, His106, Arg114, Asp142 and Tyr200, are adjusted for substrate binding and product release during catalysis. Among them, Tyr200 is especially crucial for product release. An additional potential THF binding site is identified in the cavity between two zNt-FDH molecules, which might contribute to the properties of product inhibition and THF storage reported for FDH. Together with mutagenesis studies and activity assays, the structures of zNt-FDH and its complexes provide a coherent picture of the active site and a potential THF binding site of zNt-FDH along with the substrate and product specificity, lending new insights into the molecular mechanism underlying the enzymatic properties of Nt-FDH.

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.

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