Substrate interaction dynamics and oxygen control in the active site of thymidylate synthase ThyX

2014 ◽  
Vol 459 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Hubert F. Becker ◽  
Kamel Djaout ◽  
Isabelle Lamarre ◽  
Jonathan E. Ulmer ◽  
Delphine Schaming ◽  
...  
Keyword(s):  
Thymidylate Synthase ◽  
Active Site ◽  
Oxidase Activity ◽  
Pathogenic Bacteria ◽  
Oxygen Control ◽  
Time Resolved ◽  
Time Resolved Spectroscopy ◽  
Substrate Interaction ◽  
Interaction Dynamics

Thymidylate synthase ThyX is required for synthesis of thymine in many pathogenic bacteria. Using time-resolved spectroscopy of the flavin cofactor, we unravelled the interplay between the three native substrates and show how harmful oxidase activity is avoided in aerobic environments.

scholarly journals An enzymatic activation of formaldehyde for nucleotide methylation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Charles Bou-Nader ◽  
Frederick W. Stull ◽  
Ludovic Pecqueur ◽  
Philippe Simon ◽  
Vincent Guérineau ◽  
...  
Keyword(s):  
Amino Acids ◽  
Thymidylate Synthase ◽  
Active Site ◽  
De Novo ◽  
Crystallographic Structure ◽  
De Novo Synthesis ◽  
Active Site Residues ◽  
Enzymatic Activation ◽  
Enzyme Cofactors ◽  
Unique Ability

AbstractFolate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.

scholarly journals Photophysical properties and fluorosolvatochromism of D–π–A thiophene based derivatives

RSC Advances ◽  
2020 ◽  
Vol 10 (71) ◽  
pp. 43459-43471
Author(s):  
Hussain A. Z. Sabek ◽  
Ahmed M. M. Alazaly ◽  
Dina Salah ◽  
Hesham S. Abdel-Samad ◽  
Mohamed A. Ismail ◽  
...  
Keyword(s):  
Photophysical Properties ◽  
Fluorescence Emission ◽  
Time Resolved ◽  
Time Resolved Spectroscopy ◽  
Solvation Models

Solvation-dependent photophysical properties of two push–pull thiophene-based compounds with donor–π–acceptor (D–π–A) structures were investigated using absorption, fluorescence emission and time resolved spectroscopy, and supported by different solvation models.

scholarly journals Structural and functional insights into esterase-mediated macrolide resistance

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Michał Zieliński ◽  
Jaeok Park ◽  
Barry Sleno ◽  
Albert M. Berghuis
Keyword(s):  
Active Site ◽  
Pathogenic Bacteria ◽  
Catalytic Mechanism ◽  
Three Dimensional ◽  
Resistance Mechanisms ◽  
Docking Studies ◽  
Macrolide Resistance ◽  
Flexible Docking ◽  
Sequence Identity ◽  
Substrate Spectrum

AbstractMacrolides are a class of antibiotics widely used in both medicine and agriculture. Unsurprisingly, as a consequence of their exensive usage a plethora of resistance mechanisms have been encountered in pathogenic bacteria. One of these resistance mechanisms entails the enzymatic cleavage of the macrolides’ macrolactone ring by erythromycin esterases (Eres). The most frequently identified Ere enzyme is EreA, which confers resistance to the majority of clinically used macrolides. Despite the role Eres play in macrolide resistance, research into this family enzymes has been sparse. Here, we report the first three-dimensional structures of an erythromycin esterase, EreC. EreC is an extremely close homologue of EreA, displaying more than 90% sequence identity. Two structures of this enzyme, in conjunction with in silico flexible docking studies and previously reported mutagenesis data allowed for the proposal of a detailed catalytic mechanism for the Ere family of enzymes, labeling them as metal-independent hydrolases. Also presented are substrate spectrum assays for different members of the Ere family. The results from these assays together with an examination of residue conservation for the macrolide binding site in Eres, suggests two distinct active site archetypes within the Ere enzyme family.

Time-resolved spectroscopy of single excitons bound to Te isoelectronic pairs in ZnSe

2005 ◽  
Author(s):  
A. Muller ◽  
P. Bianucci ◽  
C.K. Shih ◽  
I.C. Robin ◽  
R. Andre ◽  
...  
Keyword(s):  
Time Resolved ◽  
Time Resolved Spectroscopy

Excited States of C70and the Intersystem Crossing Process Studied by Picosecond Time-Resolved Spectroscopy in the Visible and Near-IR Region

1996 ◽  
Vol 100 (25) ◽  
pp. 10518-10522 ◽  
Author(s):  
Akira Watanabe ◽  
Osamu Ito ◽  
Motoyuki Watanabe ◽  
Haruhisa Saito ◽  
Musubu Koishi
Keyword(s):  
Excited States ◽  
Intersystem Crossing ◽  
Time Resolved ◽  
Time Resolved Spectroscopy ◽  
Near Ir
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