scholarly journals Structural insights into the mechanism of the drastic changes in enzymatic activity of the cytochrome P450 vitamin D3hydroxylase (CYP107BR1) caused by a mutation distant from the active site

2017 ◽  
Vol 73 (5) ◽  
pp. 266-275 ◽  
Author(s):  
Yoshiaki Yasutake ◽  
Tomoshi Kameda ◽  
Tomohiro Tamura
Keyword(s):  
Vitamin D ◽  
Hydrogen Bonds ◽  
Active Site ◽  
Structural Changes ◽  
Conformational Transition ◽  
Specific Activity ◽  
Cytochromes P450 ◽  
Site Directed Mutagenesis ◽  
Highly Active ◽  
Dynamics Simulations

Cytochromes P450 (P450s) are haem-containing enzymes that catalyze medically and industrially important oxidative reactions, and many P450s have been subjected to directed evolution and site-directed mutagenesis to improve their activity and substrate specificity. Nonetheless, in most cases the mechanism that leads to drastic changes in specific activity after the introduction of an amino-acid substitution distant from the active-site pocket is unclear. Here, two crystal structures of inactive mutants of the P450 vitamin D3hydroxylase (Vdh), Vdh-F106V and Vdh-L348M, which were obtained in the course of protein-engineering experiments on Vdh, are reported. The overall structures of these mutants show an open conformation similar to that of wild-type Vdh (Vdh-WT), whereas a rearrangement of the common main-chain hydrogen bonds is observed in the CD-loop (residues 102–106), resulting in a more compactly folded CD-loop relative to that of Vdh-WT. The previously reported structures of Vdh-WT and of the highly active Vdh-T107A and Vdh-K1 mutants have a more stretched CD-loop, with partial formation of 310-helix-type hydrogen bonds, both in the open and closed states. Molecular-dynamics simulations also showed that the frequency of the 310-helix is significantly reduced in Vdh-F106V and Vdh-L348M. The closed conformation is crucial for substrate and ferredoxin binding to initiate the catalytic reaction of Vdh. Therefore, it is implied that the small local structural changes observed in this study might disrupt the conformational transition from the open to the closed state, thereby leading to a complete loss of vitamin D3hydroxylase activity.

scholarly journals Active-site serine mutants of the Streptomyces albus G β-lactamase

1991 ◽  
Vol 277 (3) ◽  
pp. 647-652 ◽  
Author(s):  
F Jacob ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Serine Residue ◽  
Wild Type Enzyme ◽  
Ph Values ◽  
Streptomyces Albus ◽  
Small Production

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

WHICH IS THE PROTON-SHUTTLE IN ISOXANTHOPTERIN DEAMINASE? QM/MM MD UNDERSTANDING

2013 ◽  
Vol 12 (08) ◽  
pp. 1341002 ◽  
Author(s):  
XIN ZHANG ◽  
MING LEI
Keyword(s):  
Crystal Structure ◽  
Molecular Dynamics ◽  
Hydrogen Bonds ◽  
Proton Transfer ◽  
Glutamic Acid ◽  
Active Site ◽  
Nucleophilic Attack ◽  
Zinc Ions ◽  
Initial Model ◽  
Dynamics Simulations

The deamination process of isoxanthopterin catalyzed by isoxanthopterin deaminase was determined using the combined QM(PM3)/MM molecular dynamics simulations. In this paper, the updated PM3 parameters were employed for zinc ions and the initial model was built up based on the crystal structure. Proton transfer and following steps have been investigated in two paths: Asp336 and His285 serve as the proton shuttle, respectively. Our simulations showed that His285 is more effective than Aap336 in proton transfer for deamination of isoxanthopterin. As hydrogen bonds between the substrate and surrounding residues play a key role in nucleophilic attack, we suggested mutating Thr195 to glutamic acid, which could enhance the hydrogen bonds and help isoxanthopterin get close to the active site. The simulations which change the substrate to pterin 6-carboxylate also performed for comparison. Our results provide reference for understanding of the mechanism of deaminase and for enhancing the deamination rate of isoxanthopterin deaminase.

Mechanism of Interaction between the Heavy Chain of Factor VIII and Thrombin.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1711-1711 ◽  
Author(s):  
Keiji Nogami ◽  
Qian Zhou ◽  
Hironao Wakabayashi ◽  
Timothy Myles ◽  
Lawrence L. Leung ◽  
...  
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Active Site ◽  
Specific Activity ◽  
Solid Phase ◽  
Factor Xa ◽  
Site Directed Mutagenesis ◽  
Marginal Effect ◽  
Thrombin Cleavage ◽  
A2 Domain

Abstract Factor VIII is activated by proteolytic cleavages catalyzed by thrombin or factor Xa. An earlier study indicated that thrombin binding within the C2 domain facilitated cleavage at Arg1689 of factor VIII light chain (Nogami et al. (2000) J. Biol. Chem. 275, 25774–25780). However, thrombin-interactive region(s) within the heavy chain involved with cleaving the A1-A2 and A2-B domainal junctions remain to be determined. Several approaches were employed to examine the interactions between factor VIII heavy chain and thrombin. Fluorescence energy transfer using acrylodan-labeled A1 or A2 subunits (fluorescence donors) and a fluorescein-labeled, Phe-Pro-Arg-chloromethyl ketone active site-modified thrombin (Fl-FPR-thrombin; fluorescence acceptor) showed that FPR-thrombin bound to the A2 subunit with an ~6-fold higher affinity (Kd =36.6 nM) compared with the A1 subunit (Kd=234 nM). Solid phase binding assays using immobilized thrombin S205A, where the active-site Ser205 was converted to Ala by site directed mutagenesis, showed that the binding affinity of A2 subunit was ~3-fold greater than that of A1 subunit. Similar solid phase assays indicated that hirudin, a ligand for anion-binding exosite I of thrombin (ABE-I), effectively blocked thrombin interaction with A1 subunit while having little if any effect on its interaction with A2 subunit. Conversely, heparin, which binds ABE-II, blocked thrombin interaction with A2 subunit while showing only a marginal effect on A1 subunit binding. To identify an interactive site for thrombin in the A2 domain, we focused on two regions containing clustered acidic residues (389GluGluGluAspTrpAsp394 and 720GluAspSerTyrGluAsp725), which are localized near the N- and C-termini of the A2 domain, respectively. SDS-PAGE analyses using isolated factor VIII heavy chain as substrate showed peptides with the sequences 373–395 and 719–740 encompassing these acidic regions, blocked thrombin cleavage at both Arg372 (A1–A2 junction) and Arg740 (A2–B junction) while a 373–385 peptide did not block either cleavage. The 373–395 and 719–740 peptides competitively inhibited A2 binding to S205A thrombin in a solid phase assay (Ki=11.5 and 12.4 μM, respectively), and quenched the fluorescence of Fl-FPR-thrombin. These data demonstrate that both A2 terminal regions support interaction with thrombin. Furthermore, a B-domainless, factor VIII double mutant D392A/D394A was constructed and possessed specific activity equivalent to a severe hemophilia phenotype (<1% compared with wild type). This mutant was resistant to cleavage at Arg740 whereas cleavage at Arg372 was not appreciably affected. Thus the low specific activity of this mutant was attributed to small C-terminal extensions on the A2 subunit that were not removed following cleavage at Arg740. However, factor Xa cleavage of the mutant at Arg740 was not affected. These data suggest the acidic region comprising residues 389–394 in factor VIII A2 domain interacts with thrombin via ABE-II of the proteinase facilitating cleavage at Arg740.

Evaluation of Luciferase Thermal Stability by Arginine Saturation in the Flexible Loops

2020 ◽  
Vol 17 (1) ◽  
pp. 30-39
Author(s):  
Farzane Kargar ◽  
Mojtaba Mortazavi ◽  
Masoud Torkzadeh-Mahani ◽  
Safa Lotfi ◽  
Shahryar Shakeri
Keyword(s):  
Thermal Stability ◽  
Structural Changes ◽  
Specific Activity ◽  
Temperature Stability ◽  
3D Models ◽  
Firefly Luciferase ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Original Activity ◽  
Luciferase Enzyme

Background: The firefly luciferase enzyme is widely used in protein engineering and diverse areas of biotechnology, but the main problem with this enzyme is low-temperature stability. Previous reports indicated that surface areas of thermostable proteins are rich in arginine, which increased their thermal stability. In this study, this aspect of thermophilic proteins evaluated by mutations of surface residues to Arg. Here, we report the construction, purification, and studying of these mutated luciferases. Methods: For mutagenesis, the QuikChange site-directed mutagenesis was used and the I108R, T156R, and N177R mutant luciferases were created. In the following, the expression and purification of wild-type and mutant luciferases were conducted and their kinetic and structural properties were analyzed. To analyze the role of these Arg in these loops, the 3D models of these mutants’ enzymes were constructed in the I-TASSER server and the exact situation of these mutants was studied by the SPDBV and PyMOL software. Results: Overall, the optimum temperature of these mutated enzymes was not changed. However, after 30 min incubation of these mutated enzymes at 30°C, the I108R, T156R, N177R, and wild-type kept the 80%, 50%, 20%, and 20% of their original activity, respectively. It should be noted that substitution of these residues by Arg preserved the specific activity of firefly luciferase. Conclusion: Based on these results, it can be concluded that T156R and N177R mutants by compacting local protein structure, increased the thermostability of luciferase. However, insertion of positively charged residues in these positions create the new hydrogen bonds that associated with a series of structural changes and confirmed by intrinsic and extrinsic fluorescence spectroscopy and homology modeling studies.

scholarly journals Elucidating the regulation of glucose tolerance through the interaction between the reaction product and active site pocket residues of a β-glucosidase from Halothermothrix orenii

2019 ◽  
Author(s):  
Sushant K Sinha ◽  
Shibashis Das ◽  
Sukanya Konar ◽  
Pradip Kr. Ghorai ◽  
Rahul Das ◽  
...  
Keyword(s):  
Glucose Tolerance ◽  
Active Site ◽  
Specific Activity ◽  
Gene Encoding ◽  
Conserved Residues ◽  
Active Site Pocket ◽  
Glucose Inhibition ◽  
Glucose Tolerant ◽  
Dynamics Simulations ◽  
Hydrolysis Of

Abstractβ-glucosidase catalyzes the hydrolysis of β-1,4 linkage between two glucose molecules in cello-oligosaccharides and is prone to inhibition by the reaction product glucose. Relieving the glucose inhibition of β-glucosidase is a significant challenge. Towards the goal of understanding how glucose interacts with β-glucosidase, we expressed in Escherichia coli, the Hore_15280 gene encoding a β-glucosidase in Halothermothrix orenii. Our results show that the enzyme is glucose tolerant, and its activity stimulated in the presence of up to 0.5 M glucose. NMR analyses show the unexpected interactions between glucose and the β-glucosidase at lower concentrations of glucose that however does not lead to enzyme inhibition. We identified non-conserved residues at the aglycone-binding and the gatekeeper site and show that increased hydrophobicity at the pocket entrance and a reduction in steric hindrances are critical towards enhanced substrate accessibility and significant improvement in activity. Analysis of structures and in combination with molecular dynamics simulations show that glucose increases the accessibility of the substrate by enhancing the structural flexibility of the active site pocket and may explain the stimulation in specific activity up to 0.5 M glucose. Such novel regulation of β-glucosidase activity by its reaction product may offer novel ways of engineering glucose tolerance.

scholarly journals Residues C123 and D58 of the 2-Methylisocitrate Lyase (PrpB) Enzyme of Salmonella enterica Are Essential for Catalysis

2003 ◽  
Vol 185 (16) ◽  
pp. 4837-4843 ◽  
Author(s):  
T. L. Grimek ◽  
H. Holden ◽  
I. Rayment ◽  
J. C. Escalante-Semerena
Keyword(s):  
Active Site ◽  
Salmonella Enterica ◽  
Specific Activity ◽  
Isocitrate Lyase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Biologically Active ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Oligomeric State ◽  
Lyase Activity

ABSTRACT The prpB gene of Salmonella enterica serovar Typhimurium LT2 encodes a protein with 2-methylisocitrate (2-MIC) lyase activity, which cleaves 2-MIC into pyruvate and succinate during the conversion of propionate to pyruvate via the 2-methylcitric acid cycle. This paper reports the isolation and kinetic characterization of wild-type and five mutant PrpB proteins. Wild-type PrpB protein had a molecular mass of approximately 32 kDa per subunit, and the biologically active enzyme was comprised of four subunits. Optimal 2-MIC lyase activity was measured at pH 7.5 and 50°C, and the reaction required Mg2+ ions; equimolar concentrations of Mn2+ ions were a poor substitute for Mg2+ (28% specific activity). Dithiothreitol (DTT) or reduced glutathione (GSH) was required for optimal activity; the role of DTT or GSH was apparently not to reduce disulfide bonds, since the disulfide-specific reducing agent Tris(2-carboxyethyl)phosphine hydrochloride failed to substitute for DTT or GSH. The Km of PrpB for 2-MIC was measured at 19 μM, with a k cat of 105 s−1. Mutations in the prpB gene were introduced by site-directed mutagenesis based on the active-site residues deemed important for catalysis in the closely related phosphoenolpyruvate mutase and isocitrate lyase enzymes. Residues D58, K121, C123, and H125 of PrpB were changed to alanine, and residue R122 was changed to lysine. Nondenaturing polyacrylamide gel electrophoresis indicated that all mutant PrpB proteins retained the same oligomeric state of the wild-type enzyme, which is known to form tetramers. The PrpBK121A, PrpBH125A, and PrpBR122K mutant proteins formed enzymes that had 1,050-, 750-, and 2-fold decreases in k cat for 2-MIC lyase activity, respectively. The PrpBD58A and PrpBC123A proteins formed tetramers that displayed no detectable 2-MIC lyase activity indicating that both of these residues are essential for catalysis. Based on the proposed mechanism of the closely related isocitrate lyases, PrpB residue C123 is proposed to serve as the active site base, and residue D58 is critical for the coordination of a required Mg2+ ion.

scholarly journals Conserved conformational hierarchy across functionally divergent glycosyltransferases of the GT-B structural superfamily as determined from microsecond molecular dynamics

2020 ◽  
Author(s):  
Carlos A. Ramirez-Mondragon ◽  
Megin E. Nguyen ◽  
Jozafina Milicaj ◽  
Frank J. Tucci ◽  
Ramaiah Muthyala ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Communication Networks ◽  
Active Site ◽  
Conformational Transition ◽  
Activation Barrier ◽  
Substrate Binding ◽  
Enzyme Dynamics ◽  
Structural Class ◽  
Ionizable Residues ◽  
Dynamics Simulations

AbstractIt has long been understood that some proteins to undergo conformational transitions enroute to the Michaelis Complex to allow chemistry. Examination of crystal structures of glycosyltransferase enzymes in the GT-B structural class reveals that the presence of ligand in the active site is necessary for the protein to crystalize in the closed conformation. Herein we describe microsecond molecular dynamics simulations of two evolutionarily unrelated glycosytransferases, HepI and GtfA. Simulations were performed using these proteins in the open and closed conformations, (respectively,) and we sought to identify the major dynamical modes and communication networks which allow conformational transition between the open and closed structures. We provide the first reported evidence (within the scope of our experimental parameters) that conformational hierarchy/directionality of the interconversion between open and closed conformations is a conserved feature of enzymes of the same structural superfamily. Additionally, residues previously identified to be important for substrate binding in HepI were shown to have strong negative correlations with non-ionizable residues distal to the active site. Mutagenesis of these residues produced mutants with altered enzymatic efficiency exhibiting lower Km values, while the kcat is effectively unchanged. The negatively correlated motions of these residues are important for substrate binding and forming the Michaelis complex, without impacting the activation barrier for catalysis. This suggests that in the bi-domain HepI, the enzyme dynamics did not impact the transition state stabilization or chemistry, but rather earlier steps along the reaction coordinate, leading to the reorganization of the active site electrostatic environment required for catalysis.

scholarly journals Deciphering the Binding Mechanism of Dexamethasone Against SARS-CoV-2 Main Protease: Computational Molecular Modelling Approach

2020 ◽  
Author(s):  
Shafi Ullah Khan ◽  
Thet.thet Htar
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bonds ◽  
Active Site ◽  
Binding Mechanism ◽  
Protein Targets ◽  
High Affinity ◽  
Main Protease ◽  
Dynamics Simulations ◽  
Covalent Inhibitor ◽  
Modelling Approach

<p>At present, there are no proven agents for the treatment of 2019 coronavirus disease (COVID-19). The available evidence has not allowed guidelines to clearly recommend any drugs outside the context of clinical trials. One of the most important SARS-CoV-2 protein targets for therapeutics is the 3C-like protease (main protease, Mpro). Here in this study we utilize the recently published 6W63 crystal structure of Mpro complexed with a non-covalent inhibitor X77. Various docking methods FRED, HYBRID, CDOCKER and LEADFINDER tools were benchmark to optimally re-dock the co-crystal ligand within the active site of SARS-COV-2 Mpro. This study was restricted to molecular docking without validation by molecular dynamics simulations. CDOCKER was found to depict the exact binding of co-crystal ligand having lowest RMSD of less than 2 A. Interactions with the SARS-COV-2 Mpro may play a key role in fighting against viruses. Dexamethasone was found to bind with a high affinity to the same sites of the SARS-COV-2 Mpro than the Remdesivir. Dexamethasone was forming six hydrogen bonds compared to the three hydrogen bonds formed by Remdesivir within the active site of SARS-COV-2 Mpro. LEU141, GLY143, HIS163, GLU166, GLN192 were the key amino acid residue of SAR-COV-2 Mpro involved in stabilizing the complex between Dexamethasone and SARS-COV-2 Mpro. The results suggest the effectiveness of Dexamethasone as potent drugs against SARS-CoV-2 since it bind tightly to its Mpro. In addition, the results also suggest that dexamethasone as top antiviral treatments option than the Remdesivir with high potential to fight the SARS-CoV-2.</p>

Peripheral insertion modulates the editing activity of the isolated CP1 domain of leucyl-tRNA synthetase

2011 ◽  
Vol 440 (2) ◽  
pp. 217-227 ◽  
Author(s):  
Ru-Juan Liu ◽  
Min Tan ◽  
Dao-Hai Du ◽  
Bei-Si Xu ◽  
Gilbert Eriani ◽  
...  
Keyword(s):  
Active Site ◽  
Trna Synthetase ◽  
Site Directed Mutagenesis ◽  
Aquifex Aeolicus ◽  
Trna Synthetases ◽  
Catalytic Function ◽  
Editing Activity ◽  
Domain Docking ◽  
Dynamics Simulations

A large insertion domain called CP1 (connective peptide 1) present in class Ia aminoacyl-tRNA synthetases is responsible for post-transfer editing. LeuRS (leucyl-tRNA synthetase) from Aquifex aeolicus and Giardia lamblia possess unique 20 and 59 amino acid insertions respectively within the CP1 that are crucial for editing activity. Crystal structures of AaLeuRS-CP1 [2.4 Å (1 Å=0.1 nm)], GlLeuRS-CP1 (2.6 Å) and the insertion deletion mutant AaLeuRS-CP1Δ20 (2.5 Å) were solved to understand the role of these insertions in editing. Both insertions are folded as peripheral motifs located on the opposite side of the proteins from the active-site entrance in the CP1 domain. Docking modelling and site-directed mutagenesis showed that the insertions do not interact with the substrates. Results of molecular dynamics simulations show that the intact CP1 is more dynamic than its mutant devoid of the insertion motif. Taken together, the data show that a peripheral insertion without a substrate-binding site or major structural role in the active site may modulate catalytic function of a protein, probably from protein dynamics regulation in two respective LeuRS CP1s. Further results from proline and glycine mutational analyses intended to reduce or increase protein flexibility are consistent with this hypothesis.

Sign in / Sign up

Export Citation Format

Share Document