The impact of active site protonation on substrate ring conformation in Melanocarpus albomyces cellobiohydrolase Cel7B

2015 ◽  
Vol 17 (26) ◽  
pp. 16947-16958 ◽  
Author(s):  
Timothy C. Schutt ◽  
Vivek S. Bharadwaj ◽  
David M. Granum ◽  
C. Mark Maupin
Keyword(s):  
Active Site ◽  
Liquid Fuel ◽  
Value Added ◽  
Economic Viability ◽  
Active Site Residues ◽  
Protonation State ◽  
Ring Conformation ◽  
Melanocarpus Albomyces ◽  
Substrate Conformation ◽  
The Impact

Understanding how the protonation state of active site residues impacts the enzyme's structure and substrate conformation is important for improving the efficiency and economic viability of the degradation of cellulosic materials as feedstock for liquid fuel and value-added chemicals.

scholarly journals Exploiting correlated molecular-dynamics networks to counteract enzyme activity–stability trade-off

2018 ◽  
Vol 115 (52) ◽  
pp. E12192-E12200 ◽  
Author(s):  
Haoran Yu ◽  
Paul A. Dalby
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Active Site ◽  
Dynamic Networks ◽  
Aromatic Aldehydes ◽  
Active Site Residues ◽  
Trade Off ◽  
Highly Correlated ◽  
Dynamics Simulations ◽  
The Impact

The directed evolution of enzymes for improved activity or substrate specificity commonly leads to a trade-off in stability. We have identified an activity–stability trade-off and a loss in unfolding cooperativity for a variant (3M) of Escherichia coli transketolase (TK) engineered to accept aromatic substrates. Molecular dynamics simulations of 3M revealed increased flexibility in several interconnected active-site regions that also form part of the dimer interface. Mutating the newly flexible active-site residues to regain stability risked losing the new activity. We hypothesized that stabilizing mutations could be targeted to residues outside of the active site, whose dynamics were correlated with the newly flexible active-site residues. We previously stabilized WT TK by targeting mutations to highly flexible regions. These regions were much less flexible in 3M and would not have been selected a priori as targets using the same strategy based on flexibility alone. However, their dynamics were highly correlated with the newly flexible active-site regions of 3M. Introducing the previous mutations into 3M reestablished the WT level of stability and unfolding cooperativity, giving a 10.8-fold improved half-life at 55 °C, and increased midpoint and aggregation onset temperatures by 3 °C and 4.3 °C, respectively. Even the activity toward aromatic aldehydes increased up to threefold. Molecular dynamics simulations confirmed that the mutations rigidified the active-site via the correlated network. This work provides insights into the impact of rigidifying mutations within highly correlated dynamic networks that could also be useful for developing improved computational protein engineering strategies.

scholarly journals Crystal structure of cis-aconitate decarboxylase reveals the impact of naturally occurring human mutations on itaconate synthesis

2019 ◽  
Vol 116 (41) ◽  
pp. 20644-20654 ◽  
Author(s):  
Fangfang Chen ◽  
Peer Lukat ◽  
Azeem Ahmed Iqbal ◽  
Kyrill Saile ◽  
Volkhard Kaever ◽  
...  
Keyword(s):  
Itaconic Acid ◽  
Active Site ◽  
Disease Risk ◽  
Population Level ◽  
Therapeutic Interventions ◽  
Active Site Residues ◽  
Biotechnological Production ◽  
Naturally Occurring ◽  
The Impact

cis-Aconitate decarboxylase (CAD, also known as ACOD1 or Irg1) converts cis-aconitate to itaconate and plays central roles in linking innate immunity with metabolism and in the biotechnological production of itaconic acid by Aspergillus terreus. We have elucidated the crystal structures of human and murine CADs and compared their enzymological properties to CAD from A. terreus. Recombinant CAD is fully active in vitro without a cofactor. Murine CAD has the highest catalytic activity, whereas Aspergillus CAD is best adapted to a more acidic pH. CAD is not homologous to any known decarboxylase and appears to have evolved from prokaryotic enzymes that bind negatively charged substrates. CADs are homodimers, the active center is located in the interface between 2 distinct subdomains, and structural modeling revealed conservation in zebrafish and Aspergillus. We identified 8 active-site residues critical for CAD function and rare naturally occurring human mutations in the active site that abolished CAD activity, as well as a variant (Asn152Ser) that increased CAD activity and is common (allele frequency 20%) in African ethnicity. These results open the way for 1) assessing the potential impact of human CAD variants on disease risk at the population level, 2) developing therapeutic interventions to modify CAD activity, and 3) improving CAD efficiency for biotechnological production of itaconic acid.

The conserved active site histidine-glutamate pair of ferrochelatase coordinately catalyzes porphyrin metalation

2016 ◽  
Vol 20 (01n04) ◽  
pp. 556-569 ◽  
Author(s):  
Gregory A. Hunter ◽  
Sai Lakshmana Vankayala ◽  
Mallory E. Gillam ◽  
Fiona L. Kearns ◽  
H. Lee Woodcock ◽  
...  
Keyword(s):  
Active Site ◽  
Ferrous Iron ◽  
Metal Ion ◽  
Conformational Transition ◽  
Protoporphyrin Ix ◽  
Insertion Reaction ◽  
Active Site Residues ◽  
Protonation State ◽  
Dynamic Simulations ◽  
Ion Insertion

Ferrochelatase catalyzes the insertion of ferrous iron into protoporphyrin IX to generate heme. Despite recent research on the reaction mechanism of ferrochelatase, the precise roles and localization of individual active site residues in catalysis, particularly those involved in the insertion of the ferrous iron into the protoporphyrin IX substrate, remain controversial. One outstanding question is from which side of the macrocycle of the bound porphyin substrate is the ferrous iron substrate inserted. Pre-steady state kinetic experiments done under single-turnover conditions conclusively demonstrate that metal ion insertion is pH-dependent, and that the conserved active site His-Glu pair coordinately catalyzes the metal ion insertion reaction. Further, p[Formula: see text] calculations and molecular dynamic simulations indicate that the active site His is deprotonated and the protonation state of the Glu relates to the conformational state of ferrochelatase. Specifically, the conserved Glu in the open conformation of ferrochelatase is deprotonated, while it remains protonated in the closed conformation. These findings support not only the role of the His-Glu pair in catalyzing metal ion insertion, as these residues need to be deprotonated to bind the incoming metal ion, but also the importance of the relationship between the protonation state of the Glu residue and the conformation of ferrochelatase. Finally, the results of this study are consistent with our previous proposal that the unwinding of the [Formula: see text]-helix, the major structural determinant of the closed to open conformational transition in ferrochelatase, is associated with the Glu residue binding the Fe[Formula: see text] substrate from a mitochondrial Fe[Formula: see text] donor.

scholarly journals Histidine protonation states are key in the LigI catalytic reaction mechanism

2021 ◽  
Author(s):  
LINA ZHAO ◽  
Dibyendu Mondal ◽  
Weifeng Li ◽  
Yuguang Mu ◽  
Philipp Kaldis
Keyword(s):  
Active Site ◽  
Enzyme Catalysis ◽  
Valence Bond ◽  
Free Energy Calculations ◽  
Active Site Residues ◽  
Protonation State ◽  
Empirical Valence Bond ◽  
Hydrolysis Of ◽  
Catalytic Reaction Mechanism

Lignin is one of the world’s most abundant organic polymers, and 2-pyrone-4,6-dicarboxylate lactonase (LigI) catalyzes the hydrolysis of 2-pyrone-4,6-dicarboxylate (PDC) in the degradation of lignin. The pH has profound effects on enzyme catalysis and therefore we studied this in the context of LigI. We found that changes of the pH mostly affects surface residues, while the residues at the active site are more subject to changes of the surrounding microenvironment. In accordance with this, a high pH facilitates the deprotonation of the substrate. Detailed free energy calculations by the empirical valence bond (EVB) approach revealed that the overall hydrolysis reaction is more likely when the three active site histidines (His31, His33 and His180) are protonated at the ɛ site, however, protonation at the δ site may be favored during specific steps of reaction. Our studies have uncovered the determinant role of the protonation state of the active site residues His31, His33 and His180 in the hydrolysis of PDC.

Static and Dynamic Studies of Gasoline in View of its Octane Number and its Toxic Effect

2013 ◽  
Vol 12 (7) ◽  
pp. 451-459
Author(s):  
Ashraf Yehia El-Naggar ◽  
Mohamed A. Ebiad
Keyword(s):  
Toxic Effect ◽  
Aromatic Hydrocarbons ◽  
Octane Number ◽  
Liquid Fuel ◽  
Positive Impact ◽  
Hydrocarbon Composition ◽  
Different Temperatures ◽  
Different Seasons ◽  
The One ◽  
The Impact

Gasoline come primarily from petroleum cuts, it is the preferred liquid fuel in our lives. Two gasoline samples of octane numbers 91 and 95 from Saudi Arabia petrol stations were studied. This study was achieved at three different temperatures 20oC, 30oC and 50oC representing the change in temperatures of the different seasons of the year. Both the evaporated gases of light aromatic hydrocarbons (BTEX) of gasoline samples inside the tank were subjected to analyze qualitatively and quantitatively via capillary gas chromatography. The detailed hydrocarbon composition and the octane number of the studied gasoline samples were determined using detailed hydrocarbon analyzer. The idea of research is indicating the impact of light aromatic compounds in gasoline on the toxic effect of human and environment on the one hand, and on octane number of gasoline on the other hand. Although the value of octane number will be reduced but this will have a positive impact on the environment as a way to produce clean fuel.

Development of Xanthine Based Inhibitors Targeting Phosphodiesterase 9A

2017 ◽  
Vol 14 (10) ◽  
pp. 1122-1137 ◽  
Author(s):  
Nivedita Singh ◽  
Parameswaran Saravanan ◽  
M.S. Thakur ◽  
Sanjukta Patra
Keyword(s):  
Free Energy ◽  
Active Site ◽  
Signal Transduction Pathway ◽  
Docking Study ◽  
Primary Objective ◽  
Cgmp Level ◽  
Active Site Residues ◽  
Aromatic Fragment ◽  
Docking Approach ◽  
Free Energy Of Binding

Background: Phosphodiesterases 9A (PDE9A) is one of the prominent regulating enzymes of the signal transduction pathway having highest catalytic affinity for second messenger, cGMP. When the cGMP level is lowered, an uncontrolled expression of PDE9A may lead to various neurodegenerative diseases. To regulate the catalytic activity of PDE9A, potent inhibitors are needed. Objective: The primary objective of the present study was to develop new xanthine based inhibitors targeting PDE9A. This study was an attempt to bring structural diversification in PDE9A inhibitor development because most of the existing inhibitors are constructed over pyrazolopyrimidinone scaffold. Methods: Manual designing and parallel molecular docking approach were used for the development of xanthine derivatives. In this study, N1, N3, N9 and C8 positions of xanthine scaffold were selected as substitution sites to design 200 new compounds. Reverse docking and pharmaceutical analyses were used for final validation of most promising compounds. Results: By keeping free energy of binding cut-off of -6.0 kcal/mol, 52 compounds were screened. The compounds with substitution at N1, N3 and C8 positions of xanthine showed good occupancy in PDE9A active site pocket with a significant interaction pattern. This was further validated by screening different factors such as free energy of binding, inhibition constant and interacting active site residues in the 5Å region. Substitution at C8 position with phenyl substituent determined the inhibition affinity of compounds towards PDE9A by establishing a strong hydrophobic - hydrophobic interaction. The alkyl chain at N1 position generated selectivity of compounds towards PDE9A. The aromatic fragment at N3 position increased the binding affinity of compounds. Thus, by comparative docking study, it was found that compound 39-42 formed selective interaction towards PDE9A over other members of the PDE superfamily. Conclusion: From the present study, N1, N3 and C8 positions of xanthine were concluded as the best sites for substitution for the generation of potent PDE9A inhibitors.

Sign in / Sign up

Export Citation Format

Share Document