scholarly journals Determination of the Substrate Binding Mode to the Active Site Iron of (S)-2-Hydroxypropylphosphonic Acid Epoxidase Using17O-Enriched Substrates and Substrate Analogues†

Biochemistry ◽  
2007 ◽  
Vol 46 (44) ◽  
pp. 12628-12638 ◽  
Author(s):  
Feng Yan ◽  
Sung-Ju Moon ◽  
Pinghua Liu ◽  
Zongbao Zhao ◽  
John D. Lipscomb ◽  
...  
Keyword(s):  
Active Site ◽  
Substrate Binding ◽  
Binding Mode ◽  
Substrate Analogues

scholarly journals New insights into the mechanism of substrates trafficking in Glyoxylate/Hydroxypyruvate reductases

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Louise Lassalle ◽  
Sylvain Engilberge ◽  
Dominique Madern ◽  
Pierre Vauclare ◽  
Bruno Franzetti ◽  
...  
Keyword(s):  
Renal Failure ◽  
Active Site ◽  
Ternary Complex ◽  
Substrate Recognition ◽  
Binding Mode ◽  
X Ray ◽  
Hyperoxaluria Type

Abstract Glyoxylate accumulation within cells is highly toxic. In humans, it is associated with hyperoxaluria type 2 (PH2) leading to renal failure. The glyoxylate content within cells is regulated by the NADPH/NADH dependent glyoxylate/hydroxypyruvate reductases (GRHPR). These are highly conserved enzymes with a dual activity as they are able to reduce glyoxylate to glycolate and to convert hydroxypyruvate into D-glycerate. Despite the determination of high-resolution X-ray structures, the substrate recognition mode of this class of enzymes remains unclear. We determined the structure at 2.0 Å resolution of a thermostable GRHPR from Archaea as a ternary complex in the presence of D-glycerate and NADPH. This shows a binding mode conserved between human and archeal enzymes. We also determined the first structure of GRHPR in presence of glyoxylate at 1.40 Å resolution. This revealed the pivotal role of Leu53 and Trp138 in substrate trafficking. These residues act as gatekeepers at the entrance of a tunnel connecting the active site to protein surface. Taken together, these results allowed us to propose a general model for GRHPR mode of action.

scholarly journals The active site and substrate-binding mode of 1-aminocyclopropane-1-carboxylate oxidase determined by site-directed mutagenesis and comparative modelling studies

2004 ◽  
Vol 380 (2) ◽  
pp. 339-346 ◽  
Author(s):  
Young Sam SEO ◽  
Ahrim YOO ◽  
Jinwon JUNG ◽  
Soon-Kee SUNG ◽  
Dae Ryook YANG ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Binding Mode ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Directed Mutagenesis ◽  
Comparative Modelling

The active site and substrate-binding mode of MD-ACO1 (Malus domestica Borkh. 1-aminocyclopropane-1-carboxylate oxidase) have been determined using site-directed mutagenesis and comparative modelling methods. The MD-ACO1 protein folds into a compact jelly-roll motif comprised of eight α-helices, 12 β-strands and several long loops. The active site is well defined as a wide cleft near the C-terminus. The co-substrate ascorbate is located in cofactor Fe2+-binding pocket, the so-called ‘2-His-1-carboxylate facial triad’. In addition, our results reveal that Arg244 and Ser246 are involved in generating the reaction product during enzyme catalysis. The structure agrees well with the biochemical and site-directed mutagenesis results. The three-dimensional structure together with the steady-state kinetics of both the wild-type and mutant MD-ACO1 proteins reveal how the substrate specificity of MD-ACO1 is involved in the catalytic mechanism, providing insights into understanding the fruit ripening process at atomic resolution.

scholarly journals Effects of subsite alterations on substrate-binding mode in the active site of hen egg-white lysozyme

1993 ◽  
Vol 212 (1) ◽  
pp. 151-156 ◽  
Author(s):  
Izumi KUMAGAI ◽  
Katsumi MAENAKA ◽  
Futoshi SUNADA ◽  
Shigeki TAKEDA ◽  
Kin-ichiro MIURA
Keyword(s):  
Active Site ◽  
Substrate Binding ◽  
Binding Mode ◽  
Egg White ◽  
Hen Egg White Lysozyme ◽  
Egg White Lysozyme ◽  
Hen Egg White ◽  
Hen Egg

scholarly journals Substrate-analogue complex structure of Mycobacterium tuberculosis decaprenyl diphosphate synthase

2019 ◽  
Vol 75 (4) ◽  
pp. 212-216 ◽  
Author(s):  
Tzu-Ping Ko ◽  
Xiansha Xiao ◽  
Rey-Ting Guo ◽  
Jian-Wen Huang ◽  
Weidong Liu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Active Site ◽  
Complex Structure ◽  
Binding Mode ◽  
Farnesyl Diphosphate ◽  
The Other ◽  
Isopentenyl Diphosphate ◽  
Promising Direction ◽  
Substrate Analogues ◽  
Resolution Structure

Decaprenyl diphosphate synthase from Mycobacterium tuberculosis (MtDPPS, also known as Rv2361c) catalyzes the consecutive elongation of ω,E,Z-farnesyl diphosphate (EZ-FPP) by seven isoprene units by forming new cis double bonds. The protein folds into a butterfly-like homodimer like most other cis-type prenyltransferases. The starting allylic substrate EZ-FPP is bound to the S1 site and the homoallylic substrate to be incorporated, isopentenyl diphosphate, is bound to the S2 site. Here, a 1.55 Å resolution structure of MtDPPS in complex with the substrate analogues geranyl S-thiodiphosphate (GSPP) and isopentenyl S-thiodiphosphate bound to their respective sites in one subunit clearly shows the active-site configuration and the magnesium-coordinated geometry for catalysis. The ligand-binding mode of GSPP in the other subunit indicates a possible pathway of product translocation from the S2 site to the S1 site, as required for the next step of the reaction. The preferred binding of negatively charged effectors to the S1 site also suggests a promising direction for inhibitor design.

scholarly journals Enhancing Paraoxon Binding to Organophosphorus Hydrolase Active Site

2021 ◽  
Vol 22 (23) ◽  
pp. 12624
Author(s):  
Léa El Khoury ◽  
David L. Mobley ◽  
Dongmei Ye ◽  
Susan B. Rempe
Keyword(s):  
Binding Affinity ◽  
Active Site ◽  
Binding Free Energy ◽  
Hot Spot ◽  
Substrate Binding ◽  
Kinetic Studies ◽  
Md Simulations ◽  
Binding Mode ◽  
Free Energy Calculations ◽  
Organophosphorus Hydrolase

Organophosphorus hydrolase (OPH) is a metalloenzyme that can hydrolyze organophosphorus agents resulting in products that are generally of reduced toxicity. The best OPH substrate found to date is diethyl p-nitrophenyl phosphate (paraoxon). Most structural and kinetic studies assume that the binding orientation of paraoxon is identical to that of diethyl 4-methylbenzylphosphonate, which is the only substrate analog co-crystallized with OPH. In the current work, we used a combined docking and molecular dynamics (MD) approach to predict the likely binding mode of paraoxon. Then, we used the predicted binding mode to run MD simulations on the wild type (WT) OPH complexed with paraoxon, and OPH mutants complexed with paraoxon. Additionally, we identified three hot-spot residues (D253, H254, and I255) involved in the stability of the OPH active site. We then experimentally assayed single and double mutants involving these residues for paraoxon binding affinity. The binding free energy calculations and the experimental kinetics of the reactions between each OPH mutant and paraoxon show that mutated forms D253E, D253E-H254R, and D253E-I255G exhibit enhanced substrate binding affinity over WT OPH. Interestingly, our experimental results show that the substrate binding affinity of the double mutant D253E-H254R increased by 19-fold compared to WT OPH.

scholarly journals Active Site Architecture and Reaction Mechanism Determination of Cold Adapted β-d-galactosidase from Arthrobacter sp. 32cB

2019 ◽  
Vol 20 (17) ◽  
pp. 4301 ◽  
Author(s):  
Maria Rutkiewicz ◽  
Anna Bujacz ◽  
Marta Wanarska ◽  
Anna Wierzbicka-Wos ◽  
Hubert Cieslinski
Keyword(s):  
Reaction Mechanism ◽  
Active Site ◽  
Three Dimensional ◽  
Binding Mode ◽  
Wild Form ◽  
Cold Adapted ◽  
Transglycosylation Activity ◽  
Inactive Form ◽  
Key Factor

ArthβDG is a dimeric, cold-adapted β-d-galactosidase that exhibits high hydrolytic and transglycosylation activity. A series of crystal structures of its wild form, as well as its ArthβDG_E441Q mutein complexes with ligands were obtained in order to describe the mode of its action. The ArthβDG_E441Q mutein is an inactive form of the enzyme designed to enable observation of enzyme interaction with its substrate. The resulting three-dimensional structures of complexes: ArthβDG_E441Q/LACs and ArthβDG/IPTG (ligand bound in shallow mode) and structures of complexes ArthβDG_E441Q/LACd, ArthβDG/ONPG (ligands bound in deep mode), and galactose ArthβDG/GAL and their analysis enabled structural characterization of the hydrolysis reaction mechanism. Furthermore, comparative analysis with mesophilic analogs revealed the most striking differences in catalysis mechanisms. The key role in substrate transfer from shallow to deep binding mode involves rotation of the F581 side chain. It is worth noting that the 10-aa loop restricting access to the active site in mesophilic GH2 βDGs, in ArthβDG is moved outward. This facilitates access of substrate to active site. Such a permanent exposure of the entrance to the active site may be a key factor for improved turnover rate of the cold adapted enzyme and thus a structural feature related to its cold adaptation.

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