scholarly journals Structural and mechanistic studies of human chitinase

2014 ◽  
Vol 70 (a1) ◽  
pp. C445-C445
Author(s):  
Firas Fadel ◽  
Yuguang Zhao ◽  
Alexandra Cousido-Siah ◽  
Eduardo Howard ◽  
André Mitschler ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Catalytic Domain ◽  
Catalytic Triad ◽  
Chitin Binding Domain ◽  
X Ray ◽  
Detailed Structural Analysis ◽  
Acidic Mammalian Chitinase ◽  
Hydrogen Network ◽  
First Time

Chitinases are enzymes that hydrolyze chitin, a glucosamine polymer synthesized by lower organisms for structural purposes [1]. While humans do not synthetize chitin, they express two active chitinases, Chitotriosidase (hCHIT1) and Acidic Mammalian Chitinase (hAMCase). Both enzymes attracted attention due to their upregulation in immune system disorders [2,3]. They consist of a catalytic domain of 39 kDa and a chitin binding domain, joined by a hinge. The active site shows a cluster of three conserved acidic residues, E140, D138 and D136, linked by H-bonds, where D138 and E140 are involved in the hydrolysis reaction [1,3]. To increase our knowledge on the catalytic mechanism of human chitinases, we conducted a detailed structural analysis on hCHIT1. For this, we have improved the X-ray resolution of the apo hCHIT1 catalytic domain to 1Å. We investigated the protonation state on the catalytic site and detected a double conformation of D138, one in contact with D136 and a second one in contact with E140. Our analysis revealed for the first time different protonation states for each conformation of D138 (fig1). Interestingly, our X-ray data suggest that the catalytic E140, supposed to donate a proton in the catalytic reaction, is deprotonated in the apo form. To gain insight on the proton transition pathway during the hydrolysis, we have solved the X-ray structure of hCHIT1 complexed with the substrate at 1.05 Å. In comparison with the apo form, this structure shows a rearrangement of the protonation states of the catalytic triad triggered by the binding of the substrate. Our results led us to suggest a new hydrolysis model involving changes in the hydrogen bond network of the catalytic triad accompanied by a flip of D138 towards D136. This contributes to protonate E140, which then donates the proton to the substrate. To confirm the role of the active site's hydrogen network, we are currently studying CHIT1 by neutron crystallography and quantum mechanics.

scholarly journals Defective TiO2 Core-Shell Magnetic Photocatalyst Modified with Plasmonic Nanoparticles for Visible Light-Induced Photocatalytic Activity

Catalysts ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 672 ◽  
Author(s):  
Zuzanna Bielan ◽  
Agnieszka Sulowska ◽  
Szymon Dudziak ◽  
Katarzyna Siuzdak ◽  
Jacek Ryl ◽  
...  
Keyword(s):  
Photocatalytic Activity ◽  
Photoelectron Spectroscopy ◽  
Diffuse Reflectance Spectroscopy ◽  
Phenol Degradation ◽  
Magnetic Core ◽  
Magnetic Photocatalyst ◽  
X Ray ◽  
Transmission Electron ◽  
First Time

In the presented work, for the first time, the metal-modified defective titanium(IV) oxide nanoparticles with well-defined titanium vacancies, was successfully obtained. Introducing platinum and copper nanoparticles (NPs) as surface modifiers of defective d-TiO2 significantly increased the photocatalytic activity in both UV-Vis and Vis light ranges. Moreover, metal NPs deposition on the magnetic core allowed for the effective separation and reuse of the nanometer-sized photocatalyst from the suspension after the treatment process. The obtained Fe3O4@SiO2/d-TiO2-Pt/Cu photocatalysts were characterized by X-ray diffractometry (XRD) and specific surface area (BET) measurements, UV-Vis diffuse reflectance spectroscopy (DR-UV/Vis), X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM). Further, the mechanism of phenol degradation and the role of four oxidative species (h+, e−, •OH, and •O2−) in the studied photocatalytic process were investigated.

scholarly journals Comprehensive in silico structural-functional analysis of Enterobacter GH19 class I chitinase (chiRAM) gene: cloning and heterologous expression

2021 ◽  
Author(s):  
Shahinaz M. Abady ◽  
Khaled M. Ghanem ◽  
Nevine B. Ghanem ◽  
Amira M. Embaby
Keyword(s):  
In Silico ◽  
Catalytic Domain ◽  
Catalytic Triad ◽  
Cloning And Expression ◽  
Class I ◽  
Putative Open Reading Frame ◽  
Protein Sequence Analysis ◽  
Three Dimensional Model ◽  
Protein Motif ◽  
Chitin Binding Domain

Abstract I. Background. Present study aims to clone and express the gene-encoding chitinase / GH19 family from Enterobacter sp. in E.coli with in silico sequence analyses.. II. Methods and results. The putative open reading frame of GH19 chitinase from Enterobacter sp. strain EGY1 was cloned and expressed into pGEM-T and pET-28a + vectors, respectively using a degenerate primer. The isolated nucleotide sequence (1821 bp, Genbank accession no.: MK533791.2) was translated to chiRAM protein (606 amino acids, UniProt accession no.: A0A4D6J2L9). chiRAM in silico protein sequence analysis revealed GH19 class I chitinase: N-terminus signal peptide (Met1-Ala23), catalytic domain (Val83-Glu347 & catalytic triad Glu149, Glu171, Ser218), proline-rich hinge (Pro414 -Pro450), (polycystic kidney disease protein motif (Gly 465-Ser 533), C-terminus chitin-binding domain (Ala553- Glu593), and class I conserved motifs (NYNY and AQETGG). Three dimensional model was constructed by LOMETS MODELLER, PDB template: 2dkvA (Oryza sativa L. japonica class I chitinase). Recombinant chiRAM was overexpressed as inclusion bodies (IBs) (~ 72kDa; SDS-PAGE) in 1.0 mM IPTG induced E.coli BL21 (DE3) Rosetta at room temperature, 18 hrs post induction. Optimized expression yielded active chiRAM with 1.974 U/mL ± 0.0002, on shrimp colloidal chitin (SCC), in induced E.coli BL21 (DE3) Rosetta cells growing in SB medium. LC-MS/MS identified the 72 kDa band in the soluble fraction with 52.3% coverage sequence exclusive to Enterobacter cloacae chitinase/GH19 (WP_063869339.1). III. Conclusions. Despite the successful cloning and expression of chiRAM of Enterobacter sp. in E.coli with an appreciable chitinase activity, prospective studies would focus on minimizing IBs to facilitate chiRAM purification and characterization.

scholarly journals Mechanochemical Synthesis and Structure of the Tetrahydrate and Mesoporous Anhydrous Metforminium(2+)-N,N’-1,4-Phenylenedioxalamic Acid Cocrystal Salt: The Role of Hydrogen Bonding and N→π* Charge Assisted Interactions

2020 ◽  
Author(s):  
Sayuri Chong-Canto ◽  
Efrén V. García-Báez ◽  
Francisco J. Martínez-Martínez ◽  
Ángel Ramos-Organillo ◽  
Itzia I. Padilla-Martínez
Keyword(s):  
Hydrogen Bonding ◽  
Mechanochemical Synthesis ◽  
Water Molecules ◽  
X Ray ◽  
X Ray Crystallography ◽  
Adsorption Desorption ◽  
First Time ◽  
Selective Formation ◽  
Crystallization From Solution

A new cocrystal salt of metformin, an antidiabetic drug, and N,N’-(1,4-phenylene)dioxalamic acid, was synthesized by mechanochemical synthesis, purified by crystallization from solution and characterized by single X-ray crystallography. The structure revealed a salt-type cocrystal composed of one dicationic metformin unit, two monoanionic units of the acid and four water molecules namely H2Mf(HpOXA)2∙4H2O. X-ray powder, IR, 13C-CPMAS, thermal and BET adsorption-desorption analyses were performed to elucidate the structure of the molecular and supramolecurar structure of the anhydrous microcrystalline mesoporous solid H2Mf(HpOXA)2. The results suggest that their structures, conformation and hydrogen bonding schemes are very similar between them. To the best of our knowledge, the selective formation of the monoanion HpOXA⁻, as well as its structure in the solid, is herein reported for the first time. Regular O(-)∙∙∙C(), O(-)∙∙∙N+ and bifacial O(-)∙∙∙C()∙∙∙O(-) of n→* charge-assisted interactions are herein described in H2MfA cocrystal salts which could be responsible of the interactions of metformin in biologic systems. The results, support the participation of n→* charge-assisted interactions independently, and not just as a short contact imposed by the geometric constraint due to the hydrogen bonding patterns.

scholarly journals Crystal structure of glycosyltrehalose synthase fromSulfolobus shibataeDSM5389

2018 ◽  
Vol 74 (11) ◽  
pp. 741-746
Author(s):  
Nobuo Okazaki ◽  
Michael Blaber ◽  
Ryota Kuroki ◽  
Taro Tamada
Keyword(s):  
Crystal Structure ◽  
Amino Acids ◽  
Catalytic Mechanism ◽  
Catalytic Domain ◽  
Structural Basis ◽  
Hyperthermophilic Archaeon ◽  
X Ray ◽  
X Ray Crystallography ◽  
Sulfolobus Shibatae ◽  
Glucosidic Bond

Glycosyltrehalose synthase (GTSase) converts the glucosidic bond between the last two glucose residues of amylose from an α-1,4 bond to an α-1,1 bond, generating a nonreducing glycosyl trehaloside, in the first step of the biosynthesis of trehalose. To better understand the structural basis of the catalytic mechanism, the crystal structure of GTSase from the hyperthermophilic archaeonSulfolobus shibataeDSM5389 (5389-GTSase) has been determined to 2.4 Å resolution by X-ray crystallography. The structure of 5389-GTSase can be divided into five domains. The central domain contains the (β/α)8-barrel fold that is conserved as the catalytic domain in the α-amylase family. Three invariant catalytic carboxylic amino acids in the α-amylase family are also found in GTSase at positions Asp241, Glu269 and Asp460 in the catalytic domain. The shape of the catalytic cavity and the pocket size at the bottom of the cavity correspond to the intramolecular transglycosylation mechanism proposed from previous enzymatic studies.

Growth Morphologies, Fragmentation Patterns, and Hardness in Sodium Hydrogen Urate Monohydrate

2015 ◽  
Vol 1721 ◽  
Author(s):  
A. B. Brune ◽  
W.T. Petuskey
Keyword(s):  
Morphological Data ◽  
X Ray Diffraction ◽  
X Ray ◽  
Sodium Hydrogen ◽  
Dislocation Arrays ◽  
Fragmentation Patterns ◽  
Microscopy Techniques ◽  
First Time

ABSTRACTMechanical properties and new morphological data on synthetic sodium hydrogen urate monohydrate are reported and interpreted. Crystals formed in supersaturated aqueous solutions were identified by powder x-ray diffraction. Intact grains and separate needles were examined by several microscopy techniques, some reported here for the first time. The dominant morphology was spherulite-type, comprising tapered, branched blades (needles) radiating out of a common core. The pointed blade tips were truncated by (011) planes, corresponding to hydrogen-bonded planes. Branching was at about a 5° angle or its multiples, suggesting it accommodated by dislocation arrays at the low angle boundaries, as is often seen in twinning. Vicker’s micro-hardness, extrapolated to zero porosity, was 0.90 GPa, which is greater than the hardness measured by nano-indentation. Present results are anticipated to be useful in interpreting the mechanical characteristics of the material crystallized in vivo and its action concerning gout, and affording inferences on the role of the milieu on morphologies, fragmentation, and hardness.

Effects on the amorphous Ga2O3 film surfaces by sub-IB-metal-nano-layers

MRS Advances ◽  
2019 ◽  
Vol 4 (5-6) ◽  
pp. 285-292
Author(s):  
L. I. Juárez-Amador ◽  
M. Galván-Arellano ◽  
Y. M. Hernández-Rodríguez ◽  
J. A. Andraca-Adame ◽  
G. Romero-Paredes ◽  
...  
Keyword(s):  
Room Temperature ◽  
Rf Sputtering ◽  
Sem Analysis ◽  
X Ray Diffraction ◽  
Amorphous Films ◽  
X Ray ◽  
Plasmon Frequency ◽  
First Time ◽  
Amorphous Character

AbstractThis work reports by the first time a method to control the geometry of Ga2O3 films nanocrystallites at 350 °C. The formation of controlled shaped nano-crystallites of γ-Ga2O3 from amorphous Ga2O3 films grown by RF-Sputtering at room temperature driven by nano-layers of group IB metals (Cu, Ag or Au) is studied. The reported results can be explained by the role of subsurface metal nano-layers and the non-equilibrium nature of the sputtering processes. To study the effects on the surface structure and their optical properties arrays of amorphous-Ga2O3/IB-metal/amorphous-Ga2O3 were annealed in dry N2 atmosphere at 350 °C by 50, 100 and 150 min. The experimental results can be explained by the evolution of the amorphous character of the films amorphous films towards the nanocrystalline γ-Ga2O3 phase driven by the metal nano-layer seed nature. As the annealing time was increased the transition from amorphous-Ga2O3 to the nanocrystalline γ-Ga2O3 phase was detected by X-ray diffraction analysis. The transition to the nanocrystalline γ-Ga2O3 is demonstrated by the formation of octahedral, triangle and ball shape nanocrystallites with sizes of ∼5 to 50 nm according to FE-SEM analysis. The influence of the metal nano-layer is clearly seen by the shift of the plasmon frequency resonance produced by the Ga2O3/IB-metal/Ga2O3 arrays in the region from 400 to 600 nm caused by the modification of the interface Ga2O3/IB-metal produced by the applied annealing stages.

Homology modelling and structural analysis of human arylamine N-acetyltransferase NAT1: evidence for the conservation of a cysteine protease catalytic domain and an active-site loop

2001 ◽  
Vol 356 (2) ◽  
pp. 327-334 ◽  
Author(s):  
Fernando RODRIGUES-LIMA ◽  
Claudine DELOMÉNIE ◽  
Geoffrey H. GOODFELLOW ◽  
Denis M. GRANT ◽  
Jean-Marie DUPRET
Keyword(s):  
Crystal Structure ◽  
Cysteine Protease ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Catalytic Domain ◽  
Homology Modelling ◽  
Metabolic Activation ◽  
Cysteine Proteases ◽  
Catalytic Triad ◽  
Quality Model

Arylamine N-acetyltransferases (EC 2.3.1.5) (NATs) catalyse the biotransformation of many primary arylamines, hydrazines and their N-hydroxylated metabolites, thereby playing an important role in both the detoxification and metabolic activation of numerous xenobiotics. The recently published crystal structure of the Salmonella typhimurium NAT (StNAT) revealed the existence of a cysteine protease-like (Cys-His-Asp) catalytic triad. In the present study, a three-dimensional homology model of human NAT1, based upon the crystal structure of StNAT [Sinclair, Sandy, Delgoda, Sim and Noble (2000) Nat. Struct. Biol. 7, 560–564], is demonstrated. Alignment of StNAT and NAT1, together with secondary structure predictions, have defined a consensus region (residues 29–131) in which 37% of the residues are conserved. Homology modelling provided a good quality model of the corresponding region in human NAT1. The location of the catalytic triad was found to be identical in StNAT and NAT1. Comparison of active-site structural elements revealed that a similar length loop is conserved in both species (residues 122–131 in NAT1 model and residues 122–133 in StNAT). This observation may explain the involvement of residues 125, 127 and 129 in human NAT substrate selectivity. Our model, and the fact that cysteine protease inhibitors do not affect the activity of NAT1, suggests that human NATs may have adapted a common catalytic mechanism from cysteine proteases to accommodate it for acetyl-transfer reactions.

scholarly journals Conformational flexibility in the catalytic triad revealed by the high-resolution crystal structure ofStreptomyces erythraeustrypsin in an unliganded state

2014 ◽  
Vol 70 (3) ◽  
pp. 833-840 ◽  
Author(s):  
Elise Blankenship ◽  
Krishna Vukoti ◽  
Masaru Miyagi ◽  
David T. Lodowski
Keyword(s):  
Serine Proteases ◽  
Catalytic Mechanism ◽  
Data Bank ◽  
Catalytic Triad ◽  
Detailed Structural Analysis ◽  
Structural Rigidity ◽  
Biochemical Studies ◽  
Crystallographic Structures ◽  
Extreme Stability ◽  
Resolution Structure

With more than 500 crystal structures determined, serine proteases make up greater than one-third of all proteases structurally examined to date, making them among the best biochemically and structurally characterized enzymes. Despite the numerous crystallographic and biochemical studies of trypsin and related serine proteases, there are still considerable shortcomings in the understanding of their catalytic mechanism.Streptomyces erythraeustrypsin (SET) does not exhibit autolysis and crystallizes readily at physiological pH; hence, it is well suited for structural studies aimed at extending the understanding of the catalytic mechanism of serine proteases. While X-ray crystallographic structures of this enzyme have been reported, no coordinates have ever been made available in the Protein Data Bank. Based on this, and observations on the extreme stability and unique properties of this particular trypsin, it was decided to crystallize it and determine its structure. Here, the first sub-angstrom resolution structure of an unmodified, unliganded trypsin crystallized at physiological pH is reported. Detailed structural analysis reveals the geometry and structural rigidity of the catalytic triad in the unoccupied active site and comparison to related serine proteases provides a context for interpretation of biochemical studies of catalytic mechanism and activity.

scholarly journals Structural Insights into the Catalytic Mechanism of Granzyme B upon Substrate and Inhibitor Binding

2021 ◽  
Author(s):  
Neha Tripathi ◽  
Richard Danger ◽  
Mélanie Chesneau ◽  
Sophie Brouard ◽  
Adèle Laurent
Keyword(s):  
Active Site ◽  
Serine Proteases ◽  
Catalytic Mechanism ◽  
Granzyme B ◽  
Salt Bridge ◽  
Catalytic Triad ◽  
Catalytic Cycle ◽  
Major Step ◽  
Dynamics Simulations ◽  
First Time

Human granzyme B (hGzmB), which is present in various immune cells, has attracted much attention due to its role in various pathophysiological conditions. The hGzmB activity is triggered at a catalytic triad (His59, Asp103, Ser198), cleaving its specific substrates. To date, the drug design strategy against hGzmB mainly targets the catalytic triad, which causes the non-specificity problem of inhibitors due to the highly conserved active site in serine proteases. In the present work, microsecond classical molecular dynamics simulations are devoted to exploring the structural dynamics of the hGzmB catalytic cycle in the presence of Ac-IEPD-AMC, a known substrate (active hGzmB), and Ac-IEPD-CHO, a known inhibitor (inactive hGzmB). By comparing active and inactive forms of hGzmB in the six different stages of the hGzmB catalytic cycle, we revealed, for the very first time, an additional network of interactions involving Arg216, a residue located outside the conventional binding site. Upon activation, the His59∙∙∙Asp103 hydrogen bond is broken due to the formation of the Asp103∙∙∙Arg216 salt bridge, expanding the active site to facilitate the substrate-binding. On the contrary, the binding of inhibitor Ac-IEPD-CHO to hGzmB prevents the Arg216-mediated interactions within the catalytic triad, thus preventing hGzmB activity. In silico Arg216Ala mutation confirms the role of Arg216 in enzyme activity, as the substrate Ac-IEPD-AMC failed to bind to the mutated hGzmB. Importantly, as Arg216 is not conserved amongst the various granzymes, the current findings can be a major step to guide the design of hGzmB specific therapeutics.

scholarly journals New insights into the enzymatic mechanism of human chitotriosidase (CHIT1) catalytic domain by atomic resolution X-ray diffraction and hybrid QM/MM

2015 ◽  
Vol 71 (7) ◽  
pp. 1455-1470 ◽  
Author(s):  
Firas Fadel ◽  
Yuguang Zhao ◽  
Raul Cachau ◽  
Alexandra Cousido-Siah ◽  
Francesc X. Ruiz ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Catalytic Domain ◽  
Glycosyl Hydrolase ◽  
Catalytic Triad ◽  
X Ray Diffraction ◽  
Protonation State ◽  
Enzymatic Mechanism ◽  
Excess Concentration ◽  
Hydrolase Family

Chitotriosidase (CHIT1) is a human chitinase belonging to the highly conserved glycosyl hydrolase family 18 (GH18). GH18 enzymes hydrolyze chitin, anN-acetylglucosamine polymer synthesized by lower organisms for structural purposes. Recently, CHIT1 has attracted attention owing to its upregulation in immune-system disorders and as a marker of Gaucher disease. The 39 kDa catalytic domain shows a conserved cluster of three acidic residues, Glu140, Asp138 and Asp136, involved in the hydrolysis reaction. Under an excess concentration of substrate, CHIT1 and other homologues perform an additional activity, transglycosylation. To understand the catalytic mechanism of GH18 chitinases and the dual enzymatic activity, the structure and mechanism of CHIT1 were analyzed in detail. The resolution of the crystals of the catalytic domain was improved from 1.65 Å (PDB entry 1waw) to 0.95–1.10 Å for the apo and pseudo-apo forms and the complex with chitobiose, allowing the determination of the protonation states within the active site. This information was extended by hybrid quantum mechanics/molecular mechanics (QM/MM) calculations. The results suggest a new mechanism involving changes in the conformation and protonation state of the catalytic triad, as well as a new role for Tyr27, providing new insights into the hydrolysis and transglycosylation activities.

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