scholarly journals Structural Coupling Throughout the Active Site Hydrogen Bond Networks of Ketosteroid Isomerase and Photoactive Yellow Protein

2018 ◽  
Vol 140 (31) ◽  
pp. 9827-9843 ◽  
Author(s):  
Margaux M. Pinney ◽  
Aditya Natarajan ◽  
Filip Yabukarski ◽  
David M. Sanchez ◽  
Fang Liu ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Active Site ◽  
Photoactive Yellow Protein ◽  
Ketosteroid Isomerase ◽  
Structural Coupling ◽  
Hydrogen Bond Networks

scholarly journals Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site

2013 ◽  
Vol 110 (28) ◽  
pp. E2552-E2561 ◽  
Author(s):  
P. A. Sigala ◽  
A. T. Fafarman ◽  
J. P. Schwans ◽  
S. D. Fried ◽  
T. D. Fenn ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Proton Transfer ◽  
Active Site ◽  
Ketosteroid Isomerase

scholarly journals Hydrogen Bond Coupling in the Ketosteroid Isomerase Active Site

Biochemistry ◽  
2009 ◽  
Vol 48 (29) ◽  
pp. 6932-6939 ◽  
Author(s):  
Paul A. Sigala ◽  
Jose M. M. Caaveiro ◽  
Dagmar Ringe ◽  
Gregory A. Petsko ◽  
Daniel Herschlag
Keyword(s):  
Hydrogen Bond ◽  
Active Site ◽  
Ketosteroid Isomerase

scholarly journals Hydrogen Bonding in the Active Site of Ketosteroid Isomerase: Electronic Inductive Effects and Hydrogen Bond Coupling

Biochemistry ◽  
2010 ◽  
Vol 49 (48) ◽  
pp. 10339-10348 ◽  
Author(s):  
Philip Hanoian ◽  
Paul A. Sigala ◽  
Daniel Herschlag ◽  
Sharon Hammes-Schiffer
Keyword(s):  
Hydrogen Bond ◽  
Hydrogen Bonding ◽  
Active Site ◽  
Ketosteroid Isomerase ◽  
Inductive Effects

scholarly journals Dissecting the paradoxical effects of hydrogen bond mutations in the ketosteroid isomerase oxyanion hole

2010 ◽  
Vol 107 (5) ◽  
pp. 1960-1965 ◽  
Author(s):  
Daniel A. Kraut ◽  
Paul A. Sigala ◽  
Timothy D. Fenn ◽  
Daniel Herschlag
Keyword(s):  
Hydrogen Bond ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Ketosteroid Isomerase ◽  
X Ray ◽  
Enzyme Active Site ◽  
Hydrogen Bonding Interactions ◽  
Rate Effects ◽  
Paradoxical Effects ◽  
Moderate Rate

The catalytic importance of enzyme active-site interactions is frequently assessed by mutating specific residues and measuring the resulting rate reductions. This approach has been used in bacterial ketosteroid isomerase to probe the energetic importance of active-site hydrogen bonds donated to the dienolate reaction intermediate. The conservative Tyr16Phe mutation impairs catalysis by 105-fold, far larger than the effects of hydrogen bond mutations in other enzymes. However, the less-conservative Tyr16Ser mutation, which also perturbs the Tyr16 hydrogen bond, results in a less-severe 102-fold rate reduction. To understand the paradoxical effects of these mutations and clarify the energetic importance of the Tyr16 hydrogen bond, we have determined the 1.6-Å resolution x-ray structure of the intermediate analogue, equilenin, bound to the Tyr16Ser mutant and measured the rate effects of mutating Tyr16 to Ser, Thr, Ala, and Gly. The nearly identical 200-fold rate reductions of these mutations, together with the 6.4-Å distance observed between the Ser16 hydroxyl and equilenin oxygens in the x-ray structure, strongly suggest that the more moderate rate effect of this mutant is not due to maintenance of a hydrogen bond from Ser at position 16. These results, additional spectroscopic observations, and prior structural studies suggest that the Tyr16Phe mutation results in unfavorable interactions with the dienolate intermediate beyond loss of a hydrogen bond, thereby exaggerating the apparent energetic benefit of the Tyr16 hydrogen bond relative to the solution reaction. These results underscore the complex energetics of hydrogen bonding interactions and site-directed mutagenesis experiments.

scholarly journals A “Deep Dive” into the SARS-Cov-2 Polymerase Assembly: Identifying Novel Allosteric Sites and Analyzing the Hydrogen Bond Networks and Correlated Dynamics

2020 ◽  
Author(s):  
Khaled Barakat ◽  
Marawan Ahmed ◽  
Yasser Tabana ◽  
Minwoo Ha
Keyword(s):  
Hydrogen Bond ◽  
Active Site ◽  
Binding Modes ◽  
Deep Dive ◽  
Recent Success ◽  
Virus Life Cycle ◽  
Hydrogen Bond Networks ◽  
Allosteric Sites ◽  
Polymerase Structure ◽  
Correlated Motion

AbstractReplication of the SARS-CoV-2 genome is a fundamental step in the virus life cycle and inhibiting the SARS-CoV2 replicase machinery has been proven recently as a promising approach in combating the virus. Despite this recent success, there are still several aspects related to the structure, function and dynamics of the CoV-2 polymerase that still need to be addressed. This includes understanding the dynamicity of the various polymerase subdomains, analyzing the hydrogen bond networks at the active site and at the template entry in the presence of water, studying the binding modes of the nucleotides at the active site, highlighting positions for acceptable nucleotides’ substitutions that can be tolerated at different positions within the nascent RNA strand, identifying possible allosteric sites within the polymerase structure and studying their correlated dynamics relative to the catalytic site. Here, we combined various cutting-edge modelling tools with the recently resolved SARS-CoV-2 cryo-EM polymerase structures to fill this gap in knowledge. Our findings provide a detailed analysis of the hydrogen bond networks at various parts of the polymerase structure and suggest possible nucleotides’ substitutions that can be tolerated by the polymerase complex. We also report here three “druggable” allosteric sites within the nsp12 RdRp that can be targeted by small molecule inhibitors. Our correlated motion analysis shows that the dynamics within one of the newly identified sites are linked to the active site, indicating that targeting this site can significantly impact the catalytic activity of the SARS-CoV-2 polymerase.

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