scholarly journals An approach to optimizing the active site in a glutathione transferase by evolution in vitro

1999 ◽  
Vol 344 (1) ◽  
pp. 93-100 ◽  
Author(s):  
Lars O. HANSSON ◽  
Mikael WIDERSTEN ◽  
Bengt MANNERVIK
Keyword(s):  
Active Site ◽  
Glutathione Transferase ◽  
Single Point ◽  
Active Form ◽  
Catalytic Efficiency ◽  
Order Rate Constant ◽  
Single Point Mutation ◽  
Transition State Analogue ◽  
Active Site Mutants

A glutathione transferase (GST) mutant with four active-site substitutions (Phe10 → Pro/Ala12 → Trp/Leu107 → Phe/Leu108 → Arg) (C36) was isolated from a library of active-site mutants of human GST A1-1 by the combination of phage display and mechanism-based affinity adsorption [Hansson, Widersten and Mannervik (1997) Biochemistry 36, 11252-11260]. C36 was selected on the basis of its affinity for the transition-state analogue 1-(S-glutathionyl)-2,4,6-trinitrocyclohexadienate. C36 affords a 105-fold rate enhancement over the uncatalysed reaction between reduced glutathione and 1-chloro-2,4-dinitrobenzene (CDNB), as evidenced by the ratio between kcat/Km and the second-order rate constant k2. The present study shows that C36 can evolve to an even higher catalytic efficiency by an additional site-specific mutation. Random mutations of the fifth active-site residue 208 allowed the identification of 18 variants, of which the mutant C36 Met208 → Cys proved to be the most active form. The altered activity was substrate selective such that the catalytic efficiency with CDNB and with 1-chloro-6-trifluoromethyl-2,4-dinitrobenzene were increased 2-3-fold, whereas the activity with ethacrynic acid was decreased by a factor of 8. The results show that a single-point mutation in the active site of an enzyme may modulate the catalytic activity without being directly involved as a functional group in the enzymic mechanism. Such limited modifications are relevant both to the natural evolution and the in vitro redesign of proteins for novel functions.

Shifting Substrate Specificity of Human Glutathione Transferase (from Class Pi to Class Alpha) by a Single Point Mutation

1998 ◽  
Vol 252 (1) ◽  
pp. 184-189 ◽  
Author(s):  
Marzia Nuccetelli ◽  
Anna P. Mazzetti ◽  
Jamie Rossjohn ◽  
Michael W. Parker ◽  
Philip Board ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Point Mutation ◽  
Glutathione Transferase ◽  
Single Point ◽  
Single Point Mutation

scholarly journals Sulfated non-anticoagulant heparin derivative modifies intracellular hemoglobin, inhibits cell sickling in vitro, and prolongs survival of sickle cell mice under hypoxia

Haematologica ◽  
2021 ◽  
Author(s):  
Osheiza Abdulmalik ◽  
Noureldien H. E. Darwish ◽  
Vandhana Muralidharan-Chari ◽  
Maii Abu Taleb ◽  
Shaker A. Mousa
Keyword(s):  
Sickle Cell ◽  
Inflammatory Responses ◽  
Anticoagulant Activity ◽  
Single Point ◽  
Bleeding Complications ◽  
Single Point Mutation ◽  
Repeated Dosing ◽  
Heparin Derivative

Sickle cell disease (SCD) is an autosomal recessive genetic disease caused by a single point mutation, resulting in abnormal sickle hemoglobin (HbS). During hypoxia or dehydration, HbS polymerizes to form insoluble aggregates and induces sickling of red blood cells (RBCs). RBC sickling increases adhesiveness of RBCs to alter the rheological properties of the blood and triggers inflammatory responses, leading to hemolysis and vaso-occlusive crisis sequelae. Unfractionated heparin (UFH) and low-molecular weight heparins (LMWH) have been suggested as treatments to relieve coagulation complications in SCD. However, they are associated with bleeding complications after repeated dosing. An alternative sulfated nonanticoagulant heparin derivative (S-NACH) was previously reported to have none to low systemic anticoagulant activity and no bleeding side effects, and it interfered with P-selectindependent binding of sickle cells to endothelial cells, with concomitant decrease in the levels of adhesion biomarkers in SCD mice. S-NACH has been further engineered and structurally enhanced to bind with and modify HbS to directly inhibit sickling, thus employing a multimodal approach. Here, we show that S-NACH can (i) directly engage in Schiff-base reactions with HbS to decrease RBC sickling under both normoxia and hypoxia in vitro, ii) prolong the survival of SCD mice under hypoxia, and (iii) regulate the altered steady state levels of pro- and antiinflammatory cytokines. Thus, our proof of concept in vitro and in vivo preclinical studies demonstrate that the multimodal S-NACH is a highly promising candidate for development into an improved and optimized alternative to LMWHs for the treatment of patients with SCD.

scholarly journals A Single Point Mutation Controls the Rate of Interconversion Between the g+ and g− Rotamers of the Histidine 189 χ2 Angle That Activates Bacterial Enzyme I for Catalysis

2021 ◽  
Vol 8 ◽  
Author(s):  
Jeffrey A. Purslow ◽  
Jolene N. Thimmesch ◽  
Valeria Sivo ◽  
Trang T. Nguyen ◽  
Balabhadra Khatiwada ◽  
...  
Keyword(s):  
Protein Design ◽  
Active Site ◽  
Single Point ◽  
Conformational Dynamics ◽  
Phosphorylation Site ◽  
Phosphoryl Group ◽  
Single Point Mutation ◽  
Phosphotransferase System ◽  
Function Relationship ◽  
Enzyme I

Enzyme I (EI) of the bacterial phosphotransferase system (PTS) is a master regulator of bacterial metabolism and a promising target for development of a new class of broad-spectrum antibiotics. The catalytic activity of EI is mediated by several intradomain, interdomain, and intersubunit conformational equilibria. Therefore, in addition to its relevance as a drug target, EI is also a good model for investigating the dynamics/function relationship in multidomain, oligomeric proteins. Here, we use solution NMR and protein design to investigate how the conformational dynamics occurring within the N-terminal domain (EIN) affect the activity of EI. We show that the rotameric g+-to-g− transition of the active site residue His189 χ2 angle is decoupled from the state A-to-state B transition that describes a ∼90° rigid-body rearrangement of the EIN subdomains upon transition of the full-length enzyme to its catalytically competent closed form. In addition, we engineered EIN constructs with modulated conformational dynamics by hybridizing EIN from mesophilic and thermophilic species, and used these chimeras to assess the effect of increased or decreased active site flexibility on the enzymatic activity of EI. Our results indicate that the rate of the autophosphorylation reaction catalyzed by EI is independent from the kinetics of the g+-to-g− rotameric transition that exposes the phosphorylation site on EIN to the incoming phosphoryl group. In addition, our work provides an example of how engineering of hybrid mesophilic/thermophilic chimeras can assist investigations of the dynamics/function relationship in proteins, therefore opening new possibilities in biophysics.

scholarly journals FtsZ-Dependent Elongation of a Coccoid Bacterium

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
Ana R. Pereira ◽  
Jen Hsin ◽  
Ewa Król ◽  
Andreia C. Tavares ◽  
Pierre Flores ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Shape ◽  
Single Point ◽  
Spherical Shape ◽  
Single Point Mutation ◽  
Wild Type ◽  
Dead End ◽  
Dynamics Simulations

ABSTRACT A mechanistic understanding of the determination and maintenance of the simplest bacterial cell shape, a sphere, remains elusive compared with that of more complex shapes. Cocci seem to lack a dedicated elongation machinery, and a spherical shape has been considered an evolutionary dead-end morphology, as a transition from a spherical to a rod-like shape has never been observed in bacteria. Here we show that a Staphylococcus aureus mutant (M5) expressing the ftsZ G193D allele exhibits elongated cells. Molecular dynamics simulations and in vitro studies indicate that FtsZ G193D filaments are more twisted and shorter than wild-type filaments. In vivo , M5 cell wall deposition is initiated asymmetrically, only on one side of the cell, and progresses into a helical pattern rather than into a constricting ring as in wild-type cells. This helical pattern of wall insertion leads to elongation, as in rod-shaped cells. Thus, structural flexibility of FtsZ filaments can result in an FtsZ-dependent mechanism for generating elongated cells from cocci. IMPORTANCE The mechanisms by which bacteria generate and maintain even the simplest cell shape remain an elusive but fundamental question in microbiology. In the absence of examples of coccus-to-rod transitions, the spherical shape has been suggested to be an evolutionary dead end in morphogenesis. We describe the first observation of the generation of elongated cells from truly spherical cocci, occurring in a Staphylococcus aureus mutant containing a single point mutation in its genome, in the gene encoding the bacterial tubulin homologue FtsZ. We demonstrate that FtsZ-dependent cell elongation is possible, even in the absence of dedicated elongation machinery.

In vitro Mutagenesis of Human Dihydropteridine Reductase at the Active Site and at Altered Sites Found in the Reductases of Deficient Children

Pteridines ◽  
1996 ◽  
Vol 7 (4) ◽  
pp. 123-136 ◽  
Author(s):  
Hong-Ping Zhang ◽  
Nan Yang ◽  
Wilfred L. F. Armarego
Keyword(s):  
Active Site ◽  
Enzyme Activities ◽  
General Procedure ◽  
Maximum Velocity ◽  
Order Rate Constant ◽  
Site Directed Mutagenesis ◽  
In Vitro Mutagenesis ◽  
Dihydropteridine Reductase ◽  
Proton Source

Summary A general procedure for in vitro site-directed mutagenesis of the wild-type dihydropteridine reductase gene has been used successfully to make eight mutant proteins. Five mutations were at the active site, viz Tyrl50His, Tyrl50Ser, Tyrl50Phe, Tyr150Glu and Tyrl50Lys. The proteins were expressed as glutathione S-transferase fusion proteins from which the unconjugated reductases were obtained by thrombin cleavage. The kinetic parameters of the conjugated and unconjugated reductases were measured using natural quinonoid R-7,8(6H)-dihydrobiopterin and non-natural quinonoid RS-6-methyl-7,8(6H)-dihydropterin and NADH. The kcat (maximum velocity at saturating concentrations of substrates) and kcatl Km (first order rate constant at low concentration of substrates) values show that the phenolic OH of Tyr 150 was the most likely proton source to complete the hydride reduction of the quinonoid pterin cofactor. However in the absence of a proton source at residue 150, measurable enzyme activities were observed indicating that a proton was relayed via a water molecule(s) from some neighbouring acidic amino acid residue. Three mutant dihydropteridine reductases, which were found in defective children, have been similarly attempted, viz GlylSlSer, Gly23Asp and a threonine insertion at position 123. The enzyme activities of the first two mutant reductases were consistent with the severity of the disease. The unconjugated reductase from the third mutation could not be obtained due to proteolysis but the fusion protein was enzymically active.

scholarly journals The Role of Glutathione in the Isomerization of Δ5-Androstene- 3,17-dione Catalyzed by Human Glutathione Transferase A1-1

2001 ◽  
Vol 276 (15) ◽  
pp. 11698-11704 ◽  
Author(s):  
Pär L. Pettersson ◽  
Bengt Mannervik
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Ph Dependence ◽  
Enzymatic Catalysis ◽  
Catalytic Efficiency ◽  
Protonated Form ◽  
Hydroxyl Group ◽  
Isomerization Reaction

Human glutathione transferase (GST) A1-1 efficiently catalyzes the isomerization of Δ5-androstene-3,17-dione (AD) into Δ4-androstene-3,17-dione. High activity requires glutathione, but enzymatic catalysis occurs also in the absence of this cofactor. Glutathione alone shows a limited catalytic effect.S-Alkylglutathione derivatives do not promote the reaction, and the pH dependence of the isomerization indicates that the glutathione thiolate serves as a base in the catalytic mechanism. Mutation of the active-site Tyr9into Phe significantly decreases the steady-state kinetic parameters, alters their pH dependence, and increases the pKavalue of the enzyme-bound glutathione thiol. Thus, Tyr9promotes the reaction via its phenolic hydroxyl group in protonated form. GST A2-2 has a catalytic efficiency with AD 100-fold lower than the homologous GST A1-1. Another Alpha class enzyme, GST A4-4, is 1000-fold less active than GST A1-1. The Y9F mutant of GST A1-1 is more efficient than GST A2-2 and GST A4-4, both having a glutathione cofactor and an active-site Tyr9residue. The active sites of GST A2-2 and GST A1-1 differ by only four amino acid residues, suggesting that proper orientation of AD in relation to the thiolate of glutathione is crucial for high catalytic efficiency in the isomerization reaction. The GST A1-1-catalyzed steroid isomerization provides a complement to the previously described isomerase activity of 3β-hydroxysteroid dehydrogenase.

scholarly journals Persistent Equine Arteritis Virus Infection in HeLa Cells

2008 ◽  
Vol 82 (17) ◽  
pp. 8456-8464 ◽  
Author(s):  
Jianqiang Zhang ◽  
Peter J. Timoney ◽  
N. James MacLachlan ◽  
William H. McCollum ◽  
Udeni B. R. Balasuriya
Keyword(s):  
Persistent Infection ◽  
Single Point ◽  
Equine Arteritis Virus ◽  
Structural Proteins ◽  
Single Point Mutation ◽  
Recombinant Viruses ◽  
L Cells ◽  
Passage Number ◽  
Human Cervix

ABSTRACT The horse-adapted virulent Bucyrus (VB) strain of equine arteritis virus (EAV) established persistent infection in high-passage-number human cervix cells (HeLa-H cells; passages 170 to 221) but not in low-passage-number human cervix cells (HeLa-L cells; passages 95 to 115) or in several other cell lines that were evaluated. However, virus recovered from the 80th passage of the persistently infected HeLa-H cells (HeLa-H-EAVP80) readily established persistent infection in HeLa-L cells. Comparative sequence analysis of the entire genomes of the VB and HeLa-H-EAVP80 viruses identified 16 amino acid substitutions, including 4 in the replicase (nsp1, nsp2, nsp7, and nsp9) and 12 in the structural proteins (E, GP2, GP3, GP4, and GP5). Reverse genetic studies clearly showed that substitutions in the structural proteins but not the replicase were responsible for the establishment of persistent infection in HeLa-L cells by the HeLa-H-EAVP80 virus. It was further demonstrated that recombinant viruses with substitutions in the minor structural proteins E and GP2 or GP3 and GP4 were unable to establish persistent infection in HeLa-L cells but that recombinant viruses with combined substitutions in the E (Ser53→Cys and Val55→Ala), GP2 (Leu15→Ser, Trp31→Arg, Val87→Leu, and Ala112→Thr), GP3 (Ser115→Gly and Leu135→Pro), and GP4 (Tyr4→His and Ile109→Phe) proteins or with a single point mutation in the GP5 protein (Pro98→Leu) were able to establish persistent infection in HeLa-L cells. In summary, an in vitro model of EAV persistence in cell culture was established for the first time. This system can provide a valuable model for studying virus-host cell interactions, especially virus-receptor interactions.

scholarly journals The Carboxy-Terminal Fragment of Nucleolin Interacts with the Nucleocapsid Domain of Retroviral Gag Proteins and Inhibits Virion Assembly

2000 ◽  
Vol 74 (23) ◽  
pp. 11027-11039 ◽  
Author(s):  
Eran Bacharach ◽  
Jason Gonsky ◽  
Kimona Alin ◽  
Marianna Orlova ◽  
Stephen P. Goff
Keyword(s):  
Leukemia Virus ◽  
Single Point ◽  
Host Protein ◽  
Single Point Mutation ◽  
Gag Protein ◽  
Virion Assembly ◽  
Gag Proteins ◽  
Carboxy Terminal

ABSTRACT A yeast two-hybrid screen for cellular proteins that interact with the murine leukemia virus (MuLV) Gag protein resulted in the identification of nucleolin, a host protein known to function in ribosome assembly. The interacting fusions contained the carboxy-terminal 212 amino acids of nucleolin [Nuc(212)]. The nucleocapsid (NC) portion of Gag was necessary and sufficient to mediate the binding to Nuc(212). The interaction of Gag with Nuc(212) could be demonstrated in vitro and was manifested in vivo by the NC-dependent incorporation of Nuc(212) inside MuLV virions. Overexpression of Nuc(212), but not full-length nucleolin, potently and specifically blocked MuLV virion assembly and/or release. A mutant of MuLV, selected to specifically disrupt the binding to Nuc(212), was found to be severely defective for virion assembly. This mutant harbors a single point mutation in capsid (CA) adjacent to the CA-NC junction, suggesting a role for this region in Moloney MuLV assembly. These experiments demonstrate that selection for proteins that bind assembly domain(s) can yield potent inhibitors of virion assembly. These experiments also raise the possibility that a nucleolin-Gag interaction may be involved in virion assembly.

scholarly journals Robotic QM/MM-driven maturation of antibody combining sites

2016 ◽  
Vol 2 (10) ◽  
pp. e1501695 ◽  
Author(s):  
Ivan V. Smirnov ◽  
Andrey V. Golovin ◽  
Spyros D. Chatziefthimiou ◽  
Anastasiya V. Stepanova ◽  
Yingjie Peng ◽  
...  
Keyword(s):  
Immune Response ◽  
In Vitro Selection ◽  
Single Point ◽  
Combinatorial Libraries ◽  
Single Point Mutation ◽  
Wild Type ◽  
Selection Of ◽  
Maturation Function

In vitro selection of antibodies from large repertoires of immunoglobulin (Ig) combining sites using combinatorial libraries is a powerful tool, with great potential for generating in vivo scavengers for toxins. However, addition of a maturation function is necessary to enable these selected antibodies to more closely mimic the full mammalian immune response. We approached this goal using quantum mechanics/molecular mechanics (QM/MM) calculations to achieve maturation in silico. We preselected A17, an Ig template, from a naïve library for its ability to disarm a toxic pesticide related to organophosphorus nerve agents. Virtual screening of 167,538 robotically generated mutants identified an optimum single point mutation, which experimentally boosted wild-type Ig scavenger performance by 170-fold. We validated the QM/MM predictions via kinetic analysis and crystal structures of mutant apo-A17 and covalently modified Ig, thereby identifying the displacement of one water molecule by an arginine as delivering this catalysis.

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