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.

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 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 The multilayered organization of water-soluble proteins

2021 ◽  
Author(s):  
Lincong Wang
Keyword(s):  
Active Site ◽  
Negative Charge ◽  
Three Dimensional ◽  
Water Soluble ◽  
Partial Charge ◽  
Solvent Interaction ◽  
Upper Limit ◽  
Water Soluble Protein ◽  
Monomeric Proteins

The structural analysis of proteins has focused primarily on secondary structure, three-dimensional fold and active site while whole surface has been analyzed to a lesser extent and interior has not received much attention. Here we present an analysis of both the surfaces and the interiors of a set of water-soluble monomeric proteins in terms of solvent-excluded surface (SES) and atomic partial charge. The analysis shows that the surface of a soluble monomer has a net negative charge and is much smoother than the interior. Most interestingly with regard to both atomic partial charge and SES-defined geometric property there exists a multilayered organization from the exterior to the interior of a soluble monomer. The multilayered organization is closely related to protein-solvent interaction and should be a general feature of a water-soluble protein. Particularly the multilayered organization may set an upper limit for the size of a water-soluble monomer and plays an important role in the determination of its overall shape in solution.

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.

Two and Three Dimensional Nuclear Quadrupole Resonance in the Investigation of Structure and Bonding

2000 ◽  
Vol 55 (1-2) ◽  
pp. 29-36 ◽  
Author(s):  
Ming-Yuan Liao ◽  
Raju Subramanian ◽  
Rachel L. Yung ◽  
Gerard S. Harbison
Keyword(s):  
Active Site ◽  
Three Dimensional ◽  
Coupling Constants ◽  
Bovine Erythrocyte ◽  
Coupling Tensor ◽  
Field Gradients ◽  
Quadrupole Coupling ◽  
Quadrupole Resonance ◽  
Nuclear Quadrupole

A variety of two dimensional two-and three-dimensional NQR experiments are reviewed, showing their application to the determination of field gradients for important sites in peptides and proteins, for assigning connected transitions of nuclei with spin ≥ 5/2, and for determining hexadecapolar coupling constants. The quadrupole coupling tensor for 63Cu in the active site of a protein, bovine erythrocyte (Cu, Zn) superoxide dismutase, has been measured and is compared with the results of ab initio calculations.

Investigation of Modified Bi-Layered Tube Hydroforming by Pulsating Pressure

2011 ◽  
Vol 486 ◽  
pp. 5-8 ◽  
Author(s):  
Mohsen Loh-Mousavi ◽  
Amir Masoud Mirhosseini ◽  
Ghasem Amirian
Keyword(s):  
Finite Element Method ◽  
Finite Element ◽  
Internal Pressure ◽  
Tube Hydroforming ◽  
Three Dimensional ◽  
Key Factor ◽  
Dimensional Finite Element ◽  
Three Dimensional Finite Element ◽  
Corner Filling

In recent years, tube hydroforming has been applied in automobile and airplane industries, to decrease weight. In general, the determination of internal pressure path is a key factor in improving the formability in tube hydroforming. One of the effective methods in improving the tube hydroforming is using the pulsating internal pressure path. In this research, hydroforming of bi-layered tubes under pulsating pressure in x-shaped die is simulated by means of three dimensional finite element method. Some numerical results were compared with experimental results and show good correlation. Influence of pulsating pressure on the thickness and stress distribution have been also studied. It was shown that pulsating pressure improves the formability in hydroforming of bi-layered tubes via continues and gradual removing returnable wrinkling which is caused by a low pressure in this process. In addition, effect of some key parameters such as friction and die corner filling have been investigated.

scholarly journals Reaction Mechanism of Hydroxynitrile Lyases of the α/β-Hydrolase Superfamily

2004 ◽  
Vol 279 (19) ◽  
pp. 20501-20510 ◽  
Author(s):  
Karl Gruber ◽  
Günter Gartler ◽  
Barbara Krammer ◽  
Helmut Schwab ◽  
Christoph Kratky
Keyword(s):  
Reaction Mechanism ◽  
Active Site ◽  
Three Dimensional ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Acetone Cyanohydrin ◽  
Wild Type ◽  
Three Dimensional Structure ◽  
Catalytic Function

The hydroxynitrile lyases (HNLs) fromHevea brasiliensis(HbHNL) and fromManihot esculenta(MeHNL) are both members of the α/β-hydrolase superfamily. Mechanistic proposals have been put forward in the past for both enzymes; they differed with respect to the role of the active-site lysine residue for which a catalytic function was claimed for theHeveaenzyme but denied for theManihotenzyme. We applied a freeze-quench method to prepare crystals of the complex ofHbHNL with the biological substrate acetone cyanohydrin and determined its three-dimensional structure. Site-directed mutagenesis was used to prepare the mutant K236L, which is inactive although its three-dimensional structure is similar to the wild-type enzyme. However, the structure of the K236L-acetone cyanohydrin complex shows the substrate in a different orientation from the wild-type complex. Finite difference Poisson-Boltzmann calculations show that in the absence of Lys236the catalytic base His235would be protonated at neutral pH. All of this suggests that Lys236is instrumental for catalysis in several ways,i.e.by correctly positioning the substrate, by stabilizing the negatively charged reaction product CN-, and by modulating the basicity of the catalytic base. These data complete the elucidation of the reaction mechanism of α/β-hydrolase HNLs, in which the catalytic triad acts as a general base rather than as a nucleophile; proton abstraction from the substrate is performed by the serine, and reprotonation of the product cyanide is performed by the histidine residues. Together with a threonine side chain, the active-site serine and lysine are also involved in substrate binding.

Conformation of Ribosomes from Escherichia Coli

1974 ◽  
Vol 32 ◽  
pp. 210-211
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Binding Sites ◽  
Ribosomal Proteins ◽  
Fluorescent Dyes ◽  
Protein Biosynthesis ◽  
Three Dimensional ◽  
Spatial Arrangement ◽  
Dimensional Structure ◽  
Complete Understanding ◽  
And Function

The present knowledge of the three-dimensional structure of ribosomes is far too limited to enable a complete understanding of the various roles which ribosomes play in protein biosynthesis. The spatial arrangement of proteins and ribonuclec acids in ribosomes can be analysed in many ways. Determination of binding sites for individual proteins on ribonuclec acid and locations of the mutual positions of proteins on the ribosome using labeling with fluorescent dyes, cross-linking reagents, neutron-diffraction or antibodies against ribosomal proteins seem to be most successful approaches. Structure and function of ribosomes can be correlated be depleting the complete ribosomes of some proteins to the functionally inactive core and by subsequent partial reconstitution in order to regain active ribosomal particles.

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