scholarly journals Structural study of a single-point mutant of Sulfolobus solfataricus alcohol dehydrogenase with enhanced activity

FEBS Letters ◽  
2003 ◽  
Vol 539 (1-3) ◽  
pp. 14-18 ◽  
Author(s):  
Luciana Esposito ◽  
Ilaria Bruno ◽  
Filomena Sica ◽  
Carlo Antonio Raia ◽  
Antonietta Giordano ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Structural Study ◽  
Single Point ◽  
Sulfolobus Solfataricus ◽  
Point Mutant ◽  
Enhanced Activity

scholarly journals Unraveling the stability landscape of mutations in the SARS-CoV-2 receptor-binding domain

2020 ◽  
Author(s):  
Mohamed Raef Smaoui ◽  
Hamdi Yahyaoui
Keyword(s):  
Receptor Binding ◽  
Single Point ◽  
Protein Structures ◽  
Point Mutations ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Point Mutant ◽  
The Stability ◽  
Spike Proteins

Abstract The interaction between the receptor-binding domain of the SARS-CoV-2 spike glycoprotein and the ACE2 enzyme is believed to be the entry point of the virus into various cells in the body, including the lungs, heart, liver, and kidneys. The current focus of several therapeutic design efforts explore attempts at affecting the binding interaction between the two proteins to limit the activity of the virus and disease progression. In this work, we analyze the stability of the spike protein under all possible single-point mutations in the receptor-binding domain and computationally explore mutations that can affect the binding with the ACE2 enzyme. We unravel the mutation landscape of the receptor region and assess the toxicity potential of single and multi-point mutations, generating insights for future vaccine efforts on potential mutations that might further stabilize the spike protein and increase its infectivity. We developed a tool, called SpikeMutator, to construct full atomic protein structures of the mutant spike proteins and shared a database of 3,800 single-point mutant structures. We analyzed the recent 65,000 reported spike sequences across the globe and observed the emergence of stable multi-point mutant structures. Using the landscape, we searched through 7.5 million possible 2-point mutation combinations and report that the (R355D K424E) mutation produces one of the strongest spike proteins that therapeutic efforts should investigate for the sake of developing an effective vaccine.

scholarly journals Transcriptional Regulation of the Gene Encoding an Alcohol Dehydrogenase in the Archaeon Sulfolobus solfataricus Involves Multiple Factors and Control Elements

2003 ◽  
Vol 185 (13) ◽  
pp. 3926-3934 ◽  
Author(s):  
Gabriella Fiorentino ◽  
Raffaele Cannio ◽  
Mosè Rossi ◽  
Simonetta Bartolucci
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Alcohol Dehydrogenase ◽  
Molecular Mechanisms ◽  
Environmental Changes ◽  
Sulfolobus Solfataricus ◽  
Regulatory Sequences ◽  
Mobility Shift ◽  
Tata Element ◽  
Responsive Element

ABSTRACT A transcriptionally active region has been identified in the 5′ flanking region of the alcohol dehydrogenase gene of the crenarchaeon Sulfolobus solfataricus through the evaluation of the activity of putative transcriptional regulators and the role of the region upstream of the gene under specific metabolic circumstances. Electrophoretic mobility shift assays with crude extracts revealed protein complexes that most likely contain TATA box-associated factors. When the TATA element was deleted from the region, binding sites for both DNA binding proteins, such as the small chromatin structure-modeling Sso7d and Sso10b (Alba), and transcription factors, such as the repressor Lrs14, were revealed. To understand the molecular mechanisms underlying the substrate-induced expression of the adh gene, the promoter was analyzed for the presence of cis-acting elements recognized by specific transcription factors upon exposure of the cell to benzaldehyde. Progressive dissection of the identified promoter region restricted the analysis to a minimal responsive element (PAL) located immediately upstream of the transcription factor B-responsive element-TATA element, resembling typical bacterial regulatory sequences. A benzaldehyde-activated transcription factor (Bald) that specifically binds to the PAL cis-acting element was also identified. This protein was purified from heparin-fractionated extracts of benzaldehyde-induced cells and was shown to have a molecular mass of ∼16 kDa. The correlation between S. solfataricus adh gene activation and benzaldehyde-inducible occupation of a specific DNA sequence in its promoter suggests that a molecular signaling mechanism is responsible for the switch of the aromatic aldehyde metabolism as a response to environmental changes.

Role of Tryptophan 95 in substrate specificity and structural stability of Sulfolobus solfataricus alcohol dehydrogenase

Extremophiles ◽  
2009 ◽  
Vol 13 (5) ◽  
pp. 751-761 ◽  
Author(s):  
Angela Pennacchio ◽  
Luciana Esposito ◽  
Adriana Zagari ◽  
Mosè Rossi ◽  
Carlo A. Raia
Keyword(s):  
Alcohol Dehydrogenase ◽  
Substrate Specificity ◽  
Structural Stability ◽  
Sulfolobus Solfataricus

scholarly journals K+ Conduction and Mg2+ Blockade in a Shaker Kv-Channel Single Point Mutant with an Unusually High Conductance

2012 ◽  
Vol 103 (6) ◽  
pp. 1198-1207 ◽  
Author(s):  
Cristian Moscoso ◽  
Ariela Vergara-Jaque ◽  
Valeria Márquez-Miranda ◽  
Romina V. Sepúlveda ◽  
Ignacio Valencia ◽  
...  
Keyword(s):  
Single Point ◽  
Point Mutant ◽  
Kv Channel ◽  
High Conductance

scholarly journals Expression, purification and preliminary crystallographic studies of a single-point mutant of Mos1 mariner transposase

2004 ◽  
Vol 60 (5) ◽  
pp. 962-964 ◽  
Author(s):  
Julia M. Richardson ◽  
Lei Zhang ◽  
Severine Marcos ◽  
David J. Finnegan ◽  
Marjorie M. Harding ◽  
...  
Keyword(s):  
Single Point ◽  
Point Mutant

scholarly journals An MHV macrodomain mutant predicted to lack ADP-ribose binding activity is severely attenuated, indicating multiple roles for the macrodomain in coronavirus replication

2021 ◽  
Author(s):  
Lynden S Voth ◽  
Joseph J O'Connor ◽  
Catherine M Kerr ◽  
Ethan Doerger ◽  
Nancy Schwarting ◽  
...  
Keyword(s):  
Hepatitis Virus ◽  
Single Point ◽  
Binding Activity ◽  
Multiple Roles ◽  
Structural Protein ◽  
Transcript Accumulation ◽  
Murine Hepatitis Virus ◽  
Point Mutant

All coronaviruses (CoVs) contain a macrodomain, also termed Mac1, in non-structural protein 3 (nsp3) which binds and hydrolyzes ADP-ribose covalently attached to proteins. Despite several reports demonstrating that Mac1 is a prominent virulence factor, there is still a limited understanding of its cellular roles during infection. Currently, most of the information regarding the role of CoV Mac1 during infection is based on a single point mutant of a highly conserved asparagine-to-alanine mutation, which is known to largely eliminate Mac1 ADP-ribosylhydrolase activity. To determine if Mac1 ADP-ribose binding separately contributes to CoV replication, we compared the replication of a murine hepatitis virus (MHV) Mac1 mutant predicted to dramatically reduce ADP-ribose binding, D1329A, to the previously mentioned asparagine mutant, N1347A. D1329A and N1347A both replicated poorly in bone-marrow derived macrophages (BMDMs), were inhibited by PARP enzymes, and were highly attenuated in vivo. However, D1329A was significantly more attenuated than N1347A in all cell lines tested that were susceptible to MHV infection. In addition, D1329A retained some ability to block IFN-β transcript accumulation compared to N1347A, indicating that these two mutants impacted distinct Mac1 functions. Mac1 mutants predicted to eliminate both binding and hydrolysis activities were unrecoverable, suggesting that the combined activities of Mac1 may be essential for MHV replication. We conclude that Mac1 has multiple roles in promoting the replication of MHV, and that these results provide further evidence that Mac1 could be a prominent target for anti-CoV therapeutics.

Engineering an alcohol dehydrogenase with enhanced activity and stereoselectivity toward diaryl ketones: reduction of steric hindrance and change of the stereocontrol element

2020 ◽  
Vol 10 (6) ◽  
pp. 1650-1660
Author(s):  
Kai Wu ◽  
Zhijun Yang ◽  
Xiangguo Meng ◽  
Rong Chen ◽  
Jiankun Huang ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Steric Hindrance ◽  
Enhanced Activity

Engineering an alcohol dehydrogenase with enhanced activity and stereoselectivity toward diaryl ketones: reduction of steric hindrance and change of the stereocontrol element.

Replacing the glutamate ligand in the structural zinc site of Sulfolobus solfataricus alcohol dehydrogenase with a cysteine decreases thermostability

1995 ◽  
Vol 8 (1) ◽  
pp. 31-37 ◽  
Author(s):  
Sergio Ammendola ◽  
Giuseppe Raucci ◽  
Ottaviano Incani ◽  
Antonio Mele ◽  
Anna Tramontano ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Sulfolobus Solfataricus ◽  
Zinc Site

scholarly journals Disruption of dimerization and substrate phosphorylation inhibit factor inhibiting hypoxia-inducible factor (FIH) activity

2004 ◽  
Vol 383 (3) ◽  
pp. 429-437 ◽  
Author(s):  
David E. LANCASTER ◽  
Luke A. McNEILL ◽  
Michael A. McDONOUGH ◽  
Robin T. APLIN ◽  
Kirsty S. HEWITSON ◽  
...  
Keyword(s):  
Single Point ◽  
Hypoxia Inducible Factor ◽  
Transcriptional Response ◽  
Structural Data ◽  
Dimer Interface ◽  
Point Mutant ◽  
Catalytically Active ◽  
Substrate Phosphorylation ◽  
Asparagine Residue ◽  
Inhibit Factor

HIF (hypoxia-inducible factor) is an αβ transcription factor that modulates the hypoxic response in many animals. The cellular abundance and activity of HIF-α are regulated by its post-translational hydroxylation. The hydroxylation of HIF is catalysed by PHD (prolyl hydroxylase domain) enzymes and FIH (factorinhibiting HIF), all of which are 2-oxoglutarate- and Fe(II)-dependent dioxygenases. FIH hydroxylates a conserved asparagine residue in HIF-α (Asn-803), which blocks the binding of HIF to the transcriptional co-activator p300, preventing transcription of hypoxia-regulated genes under normoxic conditions. In the present paper, we report studies on possible mechanisms for the regulation of FIH activity. Recently solved crystal structures of FIH indicate that it is homodimeric. Site-directed mutants of FIH at residues Leu-340 and Ile-344, designed to disrupt dimerization, were generated in order to examine the importance of the dimeric state in determining FIH activity. A single point mutant, L340R (Leu-340→Arg), was shown to be predominantly monomeric and to have lost catalytic activity as measured by assays monitoring 2-oxoglutarate turnover and asparagine hydroxylation. In contrast, the I344R (Ile-344→Arg) mutant was predominantly dimeric and catalytically active. The results imply that the homodimeric form of FIH is required for productive substrate binding. The structural data also revealed a hydrophobic interaction formed between FIH and a conserved leucine residue (Leu-795) on the HIF substrate, which is close to the dimer interface. A recent report has revealed that phosphorylation of Thr-796, which is adjacent to Leu-795, enhances the transcriptional response in hypoxia. Consistent with this, we show that phosphorylation of Thr-796 prevents the hydroxylation of Asn-803 by FIH.

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