Examination of a reaction intermediate in the active site of riboflavin synthase

2003 ◽  
Vol 31 (4) ◽  
pp. 278-287 ◽  
Author(s):  
Ya-Jun Zheng ◽  
Douglas B. Jordan ◽  
Der-Ing Liao
Keyword(s):  
Active Site ◽  
Reaction Intermediate

scholarly journals Structural evidence for a reaction intermediate mimic in the active site of a sulfite dehydrogenase

2020 ◽  
Vol 56 (68) ◽  
pp. 9850-9853
Author(s):  
Ahmed Djeghader ◽  
Melanie Rossotti ◽  
Saleh Abdulkarim ◽  
Frédéric Biaso ◽  
Guillaume Gerbaud ◽  
...  
Keyword(s):  
Active Site ◽  
Spectroscopic Evidence ◽  
Reaction Intermediate

We provide structural and spectroscopic evidence for a molybdenum–phosphate adduct mimicking a proposed reaction intermediate in the active site of a prokaryotic sulfite oxidizing enzyme.

scholarly journals Does the crystal structure of vanadium nitrogenase contain a reaction intermediate? Evidence from quantum refinement

2020 ◽  
Vol 25 (6) ◽  
pp. 847-861
Author(s):  
Lili Cao ◽  
Octav Caldararu ◽  
Ulf Ryde
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Bound State ◽  
Real Space ◽  
Quantum Mechanical ◽  
Storage Site ◽  
Quantum Mechanical Calculations ◽  
Reaction Intermediate ◽  
Sulphide Ions ◽  
Vanadium Nitrogenase

Abstract Recently, a crystal structure of V-nitrogenase was presented, showing that one of the µ2 sulphide ions in the active site (S2B) is replaced by a lighter atom, suggested to be NH or NH2, i.e. representing a reaction intermediate. Moreover, a sulphur atom is found 7 Å from the S2B site, suggested to represent a storage site for this ion when it is displaced. We have re-evaluated this structure with quantum refinement, i.e. standard crystallographic refinement in which the empirical restraints (employed to ensure that the final structure makes chemical sense) are replaced by more accurate quantum–mechanical calculations. This allows us to test various interpretations of the structure, employing quantum–mechanical calculations to predict the ideal structure and to use crystallographic measures like the real-space Z-score and electron-density difference maps to decide which structure fits the crystallographic raw data best. We show that the structure contains an OH−-bound state, rather than an N2-derived reaction intermediate. Moreover, the structure shows dual conformations in the active site with ~ 14% undissociated S2B ligand, but the storage site seems to be fully occupied, weakening the suggestion that it represents a storage site for the dissociated ligand. Graphic abstract

scholarly journals Structures of a human blood group glycosyltransferase in complex with a photo-activatable UDP-Gal derivative reveal two different binding conformations

2014 ◽  
Vol 70 (8) ◽  
pp. 1015-1021 ◽  
Author(s):  
René Jørgensen ◽  
Gaëlle Batot ◽  
Karin Mannerstedt ◽  
Anne Imberty ◽  
Christelle Breton ◽  
...  
Keyword(s):  
Low Temperature ◽  
Blood Group ◽  
Human Blood ◽  
Active Site ◽  
Biological Processes ◽  
Dynamic Nature ◽  
Reaction Intermediate ◽  
Dual Specificity ◽  
Intermediate States ◽  
Sequential Addition

Glycosyltransferases (GTs) catalyse the sequential addition of monosaccharides to specific acceptor molecules and play major roles in key biological processes. GTs are classified into two main families depending on the inverted or retained stereochemistry of the glycosidic bond formed during the reaction. While the mechanism of inverting enzymes is well characterized, the precise nature of retaining GTs is still a matter of much debate. In an attempt to clarify this issue, studies were initiated to identify reaction-intermediate states by using a crystallographic approach based on caged substrates. In this paper, two distinct structures of AA(Gly)B, a dual-specificity blood group synthase, are described in complex with a UDP-galactose derivative in which the O6′′ atom is protected by a 2-nitrobenzyl group. The distinct conformations of the caged substrate in both structures of the enzyme illustrate the highly dynamic nature of its active site. An attempt was also made to photolyse the caged compound at low temperature, which unfortunately is not possible without damaging the uracil group as well. These results pave the way for kinetic crystallography experiments aiming at trapping and characterizing reaction-intermediate states in the mechanism of enzymatic glycosyl transfer.

The Active-Site Arginine ofS-Adenosylmethionine Synthetase Orients the Reaction Intermediate†

Biochemistry ◽  
1998 ◽  
Vol 37 (39) ◽  
pp. 13499-13506 ◽  
Author(s):  
Robert S. Reczkowski ◽  
John C. Taylor ◽  
George D. Markham
Keyword(s):  
Active Site ◽  
Reaction Intermediate ◽  
Adenosylmethionine Synthetase

scholarly journals Mechanism and catalytic strategy of the prokaryotic-specific GTP cyclohydrolase-IB

2017 ◽  
Vol 474 (6) ◽  
pp. 1017-1039 ◽  
Author(s):  
Naduni Paranagama ◽  
Shilah A. Bonnett ◽  
Jonathan Alvarez ◽  
Amit Luthra ◽  
Boguslaw Stec ◽  
...  
Keyword(s):  
Active Site ◽  
Competitive Inhibitor ◽  
Site Directed Mutagenesis ◽  
Antibacterial Drug ◽  
Gtp Cyclohydrolase ◽  
Reaction Intermediate ◽  
Type Ia ◽  
Biochemical Analyses ◽  
Canonical Type ◽  
Antibacterial Drug Target

Guanosine 5′-triphosphate (GTP) cyclohydrolase-I (GCYH-I) catalyzes the first step in folic acid biosynthesis in bacteria and plants, biopterin biosynthesis in mammals, and the biosynthesis of 7-deazaguanosine-modified tRNA nucleosides in bacteria and archaea. The type IB GCYH (GCYH-IB) is a prokaryotic-specific enzyme found in many pathogens. GCYH-IB is structurally distinct from the canonical type IA GCYH involved in biopterin biosynthesis in humans and animals, and thus is of interest as a potential antibacterial drug target. We report kinetic and inhibition data of Neisseria gonorrhoeae GCYH-IB and two high-resolution crystal structures of the enzyme; one in complex with the reaction intermediate analog and competitive inhibitor 8-oxoguanosine 5′-triphosphate (8-oxo-GTP), and one with a tris(hydroxymethyl)aminomethane molecule bound in the active site and mimicking another reaction intermediate. Comparison with the type IA enzyme bound to 8-oxo-GTP (guanosine 5′-triphosphate) reveals an inverted mode of binding of the inhibitor ribosyl moiety and, together with site-directed mutagenesis data, shows that the two enzymes utilize different strategies for catalysis. Notably, the inhibitor interacts with a conserved active-site Cys149, and this residue is S-nitrosylated in the structures. This is the first structural characterization of a biologically S-nitrosylated bacterial protein. Mutagenesis and biochemical analyses demonstrate that Cys149 is essential for the cyclohydrolase reaction, and S-nitrosylation maintains enzyme activity, suggesting a potential role of the S-nitrosothiol in catalysis.

scholarly journals Structural and mechanistic basis for protein glutamylation by the kinase fold

2021 ◽  
Author(s):  
Adam Osinski ◽  
Miles Black ◽  
Krzysztof Pawlowski ◽  
Zhe Chen ◽  
Yang Li ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Active Site ◽  
Bond Formation ◽  
Peptide Bond ◽  
Kinase Domain ◽  
Peptide Bond Formation ◽  
Reaction Intermediate ◽  
Catalytic Loop ◽  
Family Regulation ◽  
Mechanistic Basis

The kinase domain transfers phosphate from ATP to substrates. However, the Legionella effector SidJ adopts a kinase fold yet catalyzes calmodulin (CaM)-dependent glutamylation to inactivate the SidE ubiquitin ligases. The structural and mechanistic basis in which the kinase domain catalyzes protein glutamylation is unknown. Here we present cryo-EM reconstructions of SidJ:CaM:SidE reaction intermediate complexes. We show that the kinase-like active site of SidJ adenylates an active site Glu in SidE resulting in the formation of a stable reaction intermediate complex. An insertion in the catalytic loop of the kinase domain positions the donor Glu near the acyl-adenylate for peptide bond formation. Our structural analysis led us to discover that the SidJ paralog SdjA is a glutamylase that differentially regulates the SidE-ligases during Legionella infection. Our results uncover the structural and mechanistic basis in which the kinase fold catalyzes non-ribosomal amino acid ligations and reveal an unappreciated level of SidE-family regulation.

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