scholarly journals Unlocking the hydrolytic mechanism of GH92 α-1,2-mannosidases: computation inspires using C-glycosides as Michaelis complex mimics

2021 ◽  
Author(s):  
Santiago Alonso-Gil ◽  
Kamil Parkan ◽  
Jakub Kaminský ◽  
Radek Pohl ◽  
Takatsugu Miyazaki
Keyword(s):  
Conformational Changes ◽  
Glycoside Hydrolases ◽  
Cooperative Study ◽  
Natural Substrate ◽  
Bacteroides Thetaiotaomicron ◽  
Sugar Moiety ◽  
Hydrolytic Mechanism

The conformational changes in a sugar moiety along the hydrolytic pathway are key to understand the mechanism of glycoside hydrolases (GHs) and to design new inhibitors. The two predominant itineraries for mannosidases go via OS2  B2,5  1S5 and 3S  3H4  1C4. For the CAZy family 92, the conformational itinerary was unknown. Published complexes of Bacteroides thetaiotaomicron GH92 catalyst with a S-glycoside and mannoimidazole indicate a 4C1  4H5/1S5  1S5 mechanism. However, as observed with the GH125 family, S-glycosides may not act always as good mimics of GH’s natural substrate. Here we present a cooperative study between computations and experiments where our results predict the E5  B2,5/1S5  1S5 pathway for GH92 enzymes. Furthermore, we demonstrate the Michaelis complex mimicry of a new kind of C-disaccharides, whose biochemical applicability was still a chimera.

scholarly journals Conformational changes of peptidoglycan fragments during their interactions with vancomycin

2011 ◽  
Vol 9 (3) ◽  
pp. 422-431 ◽  
Author(s):  
Rafał Ślusarz ◽  
Magdalena Ślusarz ◽  
Justyna Samaszko ◽  
Janusz Madaj
Keyword(s):  
Molecular Dynamics ◽  
Antimicrobial Activity ◽  
Molecular Dynamics Simulations ◽  
Weak Interaction ◽  
Conformational Changes ◽  
Systems Analysis ◽  
Sugar Moiety ◽  
N Terminus ◽  
Peptide Precursors ◽  
Dynamics Simulations

AbstractSix complexes of vancomycin and peptidoglycan precursors were studied via molecular dynamics simulations. The interactions between the antibiotic and peptidoglycan fragments were identified and described in detail. All six studied modifications of the peptidoglycan precursor resulted in a weakening of the interaction with vancomycin when comparing to the native D-Ala-D-Ala-terminated fragment. It was confirmed that the N-terminus of the vancomycin is directly responsible for peptidoglycan recognition and antimicrobial activity. In simulated systems, the saccharide part of the antibiotic interacts with peptide precursors, thus it could also be important for antimicrobial activity. The complex terminated with D-Lac is the only one in which there is a weak interaction with the sugar moiety in the simulated systems. Analysis of conformational changes is a major scope of this work. The lack of interactions resulting from modification of the peptidoglycan precursors (D-Lac, D-Ser or other substitution) would be counterbalanced by proper modifications of the vancomycin moiety, especially the saccharide part of vancomycin.

scholarly journals Crystal structure of the glycoside hydrolase PssZ from Listeria monocytogenes

2019 ◽  
Vol 75 (7) ◽  
pp. 501-506 ◽  
Author(s):  
Huijun Wu ◽  
Shuai Qiao ◽  
Defeng Li ◽  
Lu Guo ◽  
Meijun Zhu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Listeria Monocytogenes ◽  
Glycoside Hydrolase ◽  
Binding Pocket ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Substrate Binding Pocket ◽  
Deep Groove ◽  
Hydrolase Family ◽  
Hydrolytic Mechanism

Biofilms are microbial communities that are embedded in the extracellular matrix. The exopolysaccharide (EPS) is a key component of the biofilm matrix that maintains the structure of the biofilm and protects the bacteria from antimicrobials. Microbial glycoside hydrolases have been exploited to disrupt biofilms by breaking down EPSs. PssZ has recently been identified as a glycoside hydrolase that can disperse aggregates of Listeria monocytogenes. In this study, the crystal structure of PssZ has been determined at 1.6 Å resolution. PssZ belongs to glycoside hydrolase family 8 and adopts a classical (α/α)6-barrel fold. This architecture forms a deep groove which may serve as the substrate-binding pocket. The conserved catalytic residues (Glu72, Trp110, Asn119, Phe167, Tyr183 and Asp232) are localized at the centre of the groove. This crystal structure will help to improve the understanding of the hydrolytic mechanism of PssZ and its application as a biofilm disrupter.

scholarly journals SusD-like protein from the human gut bacterium Bacteroides thetaiotaomicron

2014 ◽  
Vol 70 (a1) ◽  
pp. C1635-C1635
Author(s):  
Marcia Chaudet ◽  
David Rose
Keyword(s):  
Structural Data ◽  
Human Colon ◽  
Glycoside Hydrolases ◽  
Carbohydrate Binding ◽  
Bacteroides Thetaiotaomicron ◽  
Disease States ◽  
Dominant Member ◽  
Irritable Bowel ◽  
Dietary Sugars ◽  
The Impact

Nutritional research is continually demonstrating the strong interaction between the human colon microbiota and a healthy digestive system 1. It has been shown that this intestinal microbial population can influence our state of health including our metabolism, nutrient production and absorption, and the development of our immune system2. These symbiotic organisms have an important role in the metabolism of human dietary carbohydrates and exist by utilizing these sugars, which are not readily digested by upstream human enzymatic mechanisms. The ability of these microbes to utilize these sugars can also impact digestive disease states that include obesity, irritable bowel disorder, colonic cancer and Type 2 diabetes3. A dominant member of this environment is the bacterium Bacteroides thetaiotaomicron and has been characterized at efficiently utilizing carbohydrates in the colon. This symbiont has been shown to have a large repertoire of proteins and enzymes that have been predicted to be strongly involved in the capture and degradation of dietary sugars. In this study we focused on assessing the impact of a specific SusD-like protein on the utilization of dietary sugars. Biochemical evidence, as well as preliminary structural data provides support that this carbohydrate binding protein is capable of binding various dietary sugars. These interactions enable this bacterium to capture sugars derived from the colon and provide an available substrate for membrane bound glycoside hydrolases. The information gathered in this study can shed light on a part of digestion that is unclear at this point in time and create a connection between diet composition and its affect on a dominant member of the gut microbiota.

scholarly journals The hydrolysis mechanism of a GH45 cellulase and its potential relation to lytic transglycosylase and expansin function

2020 ◽  
Vol 295 (14) ◽  
pp. 4477-4487 ◽  
Author(s):  
Vivek S. Bharadwaj ◽  
Brandon C. Knott ◽  
Jerry Ståhlberg ◽  
Gregg T. Beckham ◽  
Michael F. Crowley
Keyword(s):  
Catalytic Mechanism ◽  
Structural Similarity ◽  
Glycoside Hydrolases ◽  
Free Energies ◽  
Hydroxymethyl Group ◽  
Lytic Transglycosylases ◽  
Hydrolysis Mechanism ◽  
Lytic Transglycosylase ◽  
Thermodynamic Feasibility ◽  
Hydrolytic Mechanism

Family 45 glycoside hydrolases (GH45) are endoglucanases that are integral to cellulolytic secretomes, and their ability to break down cellulose has been successfully exploited in textile and detergent industries. In addition to their industrial relevance, understanding the molecular mechanism of GH45-catalyzed hydrolysis is of fundamental importance because of their structural similarity to cell wall–modifying enzymes such as bacterial lytic transglycosylases (LTs) and expansins present in bacteria, plants, and fungi. Our understanding of the catalytic itinerary of GH45s has been incomplete because a crystal structure with substrate spanning the −1 to +1 subsites is currently lacking. Here we constructed and validated a putative Michaelis complex in silico and used it to elucidate the hydrolytic mechanism in a GH45, Cel45A from the fungus Humicola insolens, via unbiased simulation approaches. These molecular simulations revealed that the solvent-exposed active-site architecture results in lack of coordination for the hydroxymethyl group of the substrate at the −1 subsite. This lack of coordination imparted mobility to the hydroxymethyl group and enabled a crucial hydrogen bond with the catalytic acid during and after the reaction. This suggests the possibility of a nonhydrolytic reaction mechanism when the catalytic base aspartic acid is missing, as is the case in some LTs (murein transglycosylase A) and expansins. We calculated reaction free energies and demonstrate the thermodynamic feasibility of the hydrolytic and nonhydrolytic reaction mechanisms. Our results provide molecular insights into the hydrolysis mechanism in HiCel45A, with possible implications for elucidating the elusive catalytic mechanism in LTs and expansins.

scholarly journals Deglycosylation of isoflavone C–glucoside puerarin by combination of two recombinant bacterial enzymes and 3–oxo–glucose

2019 ◽  
Author(s):  
Kenichi Nakamura ◽  
Shu Zhu ◽  
Katsuko Komatsu ◽  
Masao Hattori ◽  
Makoto Iwashima
Keyword(s):  
Degradation Products ◽  
Intestinal Bacteria ◽  
Glycoside Hydrolases ◽  
Flavonoid Glycosides ◽  
Intestinal Bacterium ◽  
Bacterial Enzymes ◽  
Beneficial Effects ◽  
Sugar Moiety ◽  
Dietary Nutrients ◽  
C Complex

AbstractC–Glucosides are resistant to glycoside hydrolase activity because the anomeric carbon of glucose is directly connected to aglycone via carbon-carbon bonding. A human intestinal bacterium strain PUE related to Dorea species can metabolize the isoflavone C–glucoside puerarin (daidzein 8–C–glucoside) to daidzein and glucose by more than three bacterial enzymes which have not been well-characterized. We previously reported that 3”–oxo–puerarin is an essential reaction intermediate in enzymatic puerarin degradation and characterized a bacterial enzyme of DgpB–C complex which cleaved the C–glycosidic bond in 3”–oxo–puerarin. However, the exact enzyme catalyzing the oxidation of C–3” hydroxyl in puerarin has not been identified, and the other metabolite corresponding to the precursor of D–glucose, derived from the sugar moiety in 3”–oxo–puerarin in the cleaving reaction catalyzed by the DgpB–C complex, remains unknown.In this study, we demonstrated that recombinant DgpA, a Gfo/Idh/MocA family oxidoreductase, catalyzed puerarin oxidation in the presence of 3–oxo–glucose as the hydride accepter. In addition, enzymatic C–deglycosylation of puerarin was achieved by a combination of recombinant DgpA, DgpB–C complex, and 3–oxo–glucose. Furthermore, the metabolite derived from the sugar moiety in 3”–oxo–puerarin cleaving reaction catalyzed by DgpB–C complex was characterized as 1,5–anhydro–D–erythro –hex–1–en–3–ulose, suggesting that the C–glycosidic linkage is cleaved through a β–elimination like mechanism.ImportanceOne important role of the gut microbiota is to metabolize dietary nutrients and supplements such as flavonoid glycosides. Ingested glycosides are metabolized by intestinal bacteria to more absorbable aglycones and further degradation products which show beneficial effects in humans. Although numerous glycoside hydrolases that catalyze O–deglycosylation have been reported, enzymes responsible for C–deglycosylation are still limited. In this study, we characterized enzymes involved in C–deglycosylation of puerarin from a human intestinal bacterium PUE. To our knowledge, this is the first report of the expression, purification and characterization of an oxidoreductase involved in C–glucoside degradation. This study provides new insights for the elucidation of mechanisms of enzymatic C–deglycosylation.

scholarly journals Elimination of competing hydrolysis and coupling side reactions of a cyclodextrin glucanotransferase by directed evolution

2008 ◽  
Vol 413 (3) ◽  
pp. 517-525 ◽  
Author(s):  
Ronan M. Kelly ◽  
Hans Leemhuis ◽  
Henriëtte J. Rozeboom ◽  
Niels van Oosterwijk ◽  
Bauke W. Dijkstra ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Conformational Changes ◽  
Active Site ◽  
Outer Region ◽  
Side Reaction ◽  
Hydrolytic Activity ◽  
Glycoside Hydrolases ◽  
Side Reactions ◽  
Cyclodextrin Glucanotransferase ◽  
Active Site Residues

Thermoanaerobacterium thermosulfurigenes cyclodextrin glucanotransferase primarily catalyses the formation of cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. This enzyme also possesses unusually high hydrolytic activity as a side reaction, thought to be due to partial retention of ancestral enzyme function. This side reaction is undesirable, since it produces short saccharides that are responsible for the breakdown of the cyclodextrins formed, thus limiting the yield of cyclodextrins produced. To reduce the competing hydrolysis reaction, while maintaining the cyclization activity, we applied directed evolution, introducing random mutations throughout the cgt gene by error-prone PCR. Mutations in two residues, Ser-77 and Trp-239, on the outer region of the active site, lowered the hydrolytic activity up to 15-fold with retention of cyclization activity. In contrast, mutations within the active site could not lower hydrolytic rates, indicating an evolutionary optimized role for cyclodextrin formation by residues within this region. The crystal structure of the most effective mutant, S77P, showed no alterations to the peptide backbone. However, subtle conformational changes to the side chains of active-site residues had occurred, which may explain the increased cyclization/hydrolysis ratio. This indicates that secondary effects of mutations located on the outer regions of the catalytic site are required to lower the rates of competing side reactions, while maintaining the primary catalytic function. Subsequent functional analysis of various glucanotransferases from the superfamily of glycoside hydrolases also suggests a gradual evolutionary progression of these enzymes from a common ‘intermediate-like’ ancestor towards specific transglycosylation activity.

scholarly journals Effect of Dimer Dissociation on Activity and Thermostability of the α-Glucuronidase from Geobacillus stearothermophilus: Dissecting the Different Oligomeric Forms of Family 67 Glycoside Hydrolases

2004 ◽  
Vol 186 (20) ◽  
pp. 6928-6937 ◽  
Author(s):  
Dalia Shallom ◽  
Gali Golan ◽  
Gil Shoham ◽  
Yuval Shoham
Keyword(s):  
Conformational Changes ◽  
Conformational Stability ◽  
Biological Significance ◽  
Glycoside Hydrolases ◽  
Geobacillus Stearothermophilus ◽  
Wild Type ◽  
Cellvibrio Japonicus ◽  
Hydrolyzing Enzymes ◽  
Oligomeric Forms ◽  
Active Site Region

ABSTRACT The oligomeric organization of enzymes plays an important role in many biological processes, such as allosteric regulation, conformational stability and thermal stability. α-Glucuronidases are family 67 glycosidases that cleave the α-1,2-glycosidic bond between 4-O-methyl-d-glucuronic acid and xylose units as part of an array of hemicellulose-hydrolyzing enzymes. Currently, two crystal structures of α-glucuronidases are available, those from Geobacillus stearothermophilus (AguA) and from Cellvibrio japonicus (GlcA67A). Both enzymes are homodimeric, but surprisingly their dimeric organization is different, raising questions regarding the significance of dimerization for the enzymes' activity and stability. Structural comparison of the two enzymes suggests several elements that are responsible for the different dimerization organization. Phylogenetic analysis shows that the α-glucuronidases AguA and GlcA67A can be classified into two distinct subfamilies of bacterial α-glucuronidases, where the dimer-forming residues of each enzyme are conserved only within its own subfamily. It seems that the different dimeric forms of AguA and GlcA67A represent the two alternative dimeric organizations of these subfamilies. To study the biological significance of the dimerization in α-glucuronidases, we have constructed a monomeric form of AguA by mutating three of its interface residues (W328E, R329T, and R665N). The activity of the monomer was significantly lower than the activity of the wild-type dimeric AguA, and the optimal temperature for activity of the monomer was around 35°C, compared to 65°C of the wild-type enzyme. Nevertheless, the melting temperature of the monomeric protein, 72.9°C, was almost identical to that of the wild-type, 73.4°C. It appears that the dimerization of AguA is essential for efficient catalysis and that the dissociation into monomers results in subtle conformational changes in the structure which indirectly influence the active site region and reduce the activity. Structural and mechanistic explanations for these effects are discussed.

Influence of Calcium Ions on the Plant Cell Surface: Membrane Fusions and Conformational Changes

1974 ◽  
Vol 32 ◽  
pp. 154-155
Author(s):  
D. James Morré ◽  
Charles E. Bracker ◽  
William J. VanDerWoude
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Conformational Changes ◽  
Calcium Ions ◽  
Surface Membrane ◽  
Carboxyl Groups ◽  
Cross Linking ◽  
Ultrastructural Evidence ◽  
Glycine Max L ◽  
Wall Extensibility

Calcium ions in the concentration range 5-100 mM inhibit auxin-induced cell elongation and wall extensibility of plant stems. Inhibition of wall extensibility requires that the tissue be living; growth inhibition cannot be explained on the basis of cross-linking of carboxyl groups of cell wall uronides by calcium ions. In this study, ultrastructural evidence was sought for an interaction of calcium ions with some component other than the wall at the cell surface of soybean (Glycine max (L.) Merr.) hypocotyls.

Role of spectrin in membrane fusion and in shape change of human erythrocyte ghost

1978 ◽  
Vol 36 (2) ◽  
pp. 328-329
Author(s):  
Hideo Hayashi ◽  
Yoshikazu Hirai ◽  
John T. Penniston
Keyword(s):  
Conformational Changes ◽  
Human Erythrocyte ◽  
Shape Change ◽  
Membrane Surface ◽  
Slight Modification ◽  
Active Role ◽  
Main Role ◽  
Bank Blood ◽  
Human Erythrocyte Ghost

Spectrin is a membrane associated protein most of which properties have been tentatively elucidated. A main role of the protein has been assumed to give a supporting structure to inside of the membrane. As reported previously, however, the isolated spectrin molecule underwent self assemble to form such as fibrous, meshwork, dispersed or aggregated arrangements depending upon the buffer suspended and was suggested to play an active role in the membrane conformational changes. In this study, the role of spectrin and actin was examined in terms of the molecular arrangements on the erythrocyte membrane surface with correlation to the functional states of the ghosts.Human erythrocyte ghosts were prepared from either freshly drawn or stocked bank blood by the method of Dodge et al with a slight modification as described before. Anti-spectrin antibody was raised against rabbit by injection of purified spectrin and partially purified.

Pinocytotic-Like Forms of Mitochondria From Rat Anterior Pituitary

1968 ◽  
Vol 26 ◽  
pp. 146-147
Author(s):  
Burton B. Silver
Keyword(s):  
Electron Micrographs ◽  
Conformational Changes ◽  
Anterior Pituitary ◽  
Dynamic State ◽  
Surrounding Cell ◽  
The Matrix ◽  
The Moment ◽  
Cell Medium ◽  
Sectioned Tissue ◽  
State Of Activity

Sectioned tissue rarely indicates evidence of what is probably a highly dynamic state of activity in mitochondria which have been reported to undergo a variety of movements such as streaming, divisions and coalescence. Recently, mitochondria from the rat anterior pituitary have been fixed in a variety of configurations which suggest that conformational changes were occurring at the moment of fixation. Pinocytotic-like vacuoles which may be taking in or expelling materials from the surrounding cell medium, appear to be forming in some of the mitochondria. In some cases, pores extend into the matrix of the mitochondria. In other forms, the remains of what seems to be pinched off vacuoles are evident in the mitochondrial interior. Dense materials, resembling secretory droplets, appear at the junction of the pores and the cytoplasm. The droplets are similar to the secretory materials commonly identified in electron micrographs of the anterior pituitary.

Sign in / Sign up

Export Citation Format

Share Document