scholarly journals Reverse Ordered Sequential Mechanism for Lactoperoxidase with Inhibition by Hydrogen Peroxide

Antioxidants ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1646
Author(s):  
Kellye Cupp-Sutton ◽  
Michael T. Ashby
Keyword(s):  
Hydrogen Peroxide ◽  
Antimicrobial Agent ◽  
Resting State ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Biphasic Kinetics ◽  
Biologically Relevant ◽  
Sequential Mechanism ◽  
Mucosal Glands ◽  
Ordered Sequential Mechanism

Lactoperoxidase (LPO, FeIII in its resting state in the absence of substrates)—an enzyme secreted from human mammary, salivary, and other mucosal glands—catalyzes the oxidation of thiocyanate (SCN−) by hydrogen peroxide (H2O2) to produce hypothiocyanite (OSCN−), which functions as an antimicrobial agent. The accepted catalytic mechanism, called the halogen cycle, comprises a two-electron oxidation of LPO by H2O2 to produce oxoiron(IV) radicals, followed by O-atom transfer to SCN−. However, the mechanism does not explain biphasic kinetics and inhibition by H2O2 at low concentration of reducing substrate, conditions that may be biologically relevant. We propose an ordered sequential mechanism in which the order of substrate binding is reversed, first SCN− and then H2O2. The sequence of substrate binding that is described by the halogen cycle mechanism is actually inhibitory.

Copper–oxygen Dynamics in Tyrosinase

2019 ◽  
Author(s):  
Nobutaka Fujieda ◽  
Sachiko Yanagisawa ◽  
Minoru Kubo ◽  
Genji Kurisu ◽  
Shinobu Itoh
Keyword(s):  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Active Form ◽  
Copper Ion ◽  
Atomic Resolution ◽  
Oxygen Species ◽  
X Ray ◽  
Oxygen Dynamics ◽  
Insight Into ◽  
Copper Centre

To unveil the activation of dioxygen on the copper centre (Cu<sub>2</sub>O<sub>2</sub>core) of tyrosinase, we performed X-ray crystallograpy with active-form tyrosinase at near atomic resolution. This study provided a novel insight into the catalytic mechanism of the tyrosinase, including the rearrangement of copper-oxygen species as well as the intramolecular migration of copper ion induced by substrate-binding.<br>

Structure and mechanism of the γ-glutamyl-γ-aminobutyrate hydrolase SpuA from Pseudomonas aeruginosa

2021 ◽  
Vol 77 (10) ◽  
pp. 1305-1316
Author(s):  
Yujing Chen ◽  
Haizhu Jia ◽  
Jianyu Zhang ◽  
Yakun Liang ◽  
Ruihua Liu ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Class I ◽  
Polyamine Metabolism ◽  
Binding Assays ◽  
Starting Point ◽  
Family Activity ◽  
Living Organisms

Polyamines are important regulators in all living organisms and are implicated in essential biological processes including cell growth, differentiation and apoptosis. Pseudomonas aeruginosa possesses an spuABCDEFGHI gene cluster that is involved in the metabolism and uptake of two polyamines: spermidine and putrescine. In the proposed γ-glutamylation–putrescine metabolism pathway, SpuA hydrolyzes γ-glutamyl-γ-aminobutyrate (γ-Glu-GABA) to glutamate and γ-aminobutyric acid (GABA). In this study, crystal structures of P. aeruginosa SpuA are reported, confirming it to be a member of the class I glutamine amidotransferase (GAT) family. Activity and substrate-binding assays confirm that SpuA exhibits a preference for γ-Glu-GABA as a substrate. Structures of an inactive H221N mutant were determined with bound glutamate thioester intermediate or glutamate product, thus delineating the active site and substrate-binding pocket and elucidating the catalytic mechanism. The crystal structure of another bacterial member of the class I GAT family from Mycolicibacterium smegmatis (MsGATase) in complex with glutamine was determined for comparison and reveals a binding site for glutamine. Activity assays confirm that MsGATase has activity for glutamine as a substrate but not for γ-Glu-GABA. The work reported here provides a starting point for further investigation of polyamine metabolism in P. aeruginosa.

scholarly journals Interaction Between Titanium Implant Surfaces and Hydrogen Peroxide in Biologically Relevant Environments

2004 ◽  
Vol 823 ◽  
Author(s):  
Julie Muyco ◽  
Timothy Ratto ◽  
Christine Orme ◽  
Joanna McKittrick ◽  
John Frangos
Keyword(s):  
Hydrogen Peroxide ◽  
Raman Spectroscopy ◽  
Atomic Force Microscope ◽  
Titanium Implant ◽  
Dilute Solutions ◽  
Interaction Layer ◽  
Biologically Relevant ◽  
Light Passing ◽  
Atomic Force ◽  
Force Curves

AbstractTitanium was exposed to dilute solutions of hydrogen peroxide (H2O2) to better characterize the interaction at the interface between the solution and metal. The intensity of light passing through films of known thickness of titanium on quartz was measured as a function of time in contact with H2O2in concentrations of 0.3% and 1.0%. An atomic force microscope (AFM) was used to record deflection-distance (force) curves as a probe approached the interface of titanium in contact with solution containing 0.3% of H2O2. The interaction layer measured using AFM techniques was much greater than the thickness of the titanium films used in this study. Raman spectroscopy taken during interaction shows the emergence of a Ti-peroxy gel and titania after 2 hours in contact with 0.3% H2O2solution.

scholarly journals A crystallographic study of the Toho-1 β-lactamase acylation mechanism

2014 ◽  
Vol 70 (a1) ◽  
pp. C1207-C1207
Author(s):  
Leighton Coates
Keyword(s):  
Hydrogen Bonding ◽  
Transition State ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Ligand Complex ◽  
Transition State Analog ◽  
Hydrogen Bonding Interactions ◽  
Active Site Region ◽  
Neutron Crystallography

β-lactam antibiotics have been used effectively over several decades against many types of highly virulent bacteria. The predominant cause of resistance to these antibiotics in Gram-negative bacterial pathogens is the production of serine β-lactamase enzymes. A key aspect of the class A serine β-lactamase mechanism that remains unresolved and controversial is the identity of the residue acting as the catalytic base during the acylation reaction. Multiple mechanisms have been proposed for the formation of the acyl-enzyme intermediate that are predicated on understanding the protonation states and hydrogen-bonding interactions among the important residues involved in substrate binding and catalysis of these enzymes. For resolving a controversy of this nature surrounding the catalytic mechanism, neutron crystallography is a powerful complement to X-ray crystallography that can explicitly determine the location of deuterium atoms in proteins, thereby directly revealing the hydrogen-bonding interactions of important amino acid residues. Neutron crystallography was used to unambiguously reveal the ground-state active site protonation states and the resulting hydrogen-bonding network in two ligand-free Toho-1 β-lactamase mutants which provided remarkably clear pictures of the active site region prior to substrate binding and subsequent acylation [1,2] and an acylation transition-state analog, benzothiophene-2-boronic acid (BZB), which was also isotopically enriched with 11B. The neutron structure revealed the locations of all deuterium atoms in the active site region and clearly indicated that Glu166 is protonated in the BZB transition-state analog complex. As a result, the complete hydrogen-bonding pathway throughout the active site region could then deduced for this protein-ligand complex that mimics the acylation tetrahedral intermediate [3].

scholarly journals Structures of an engineered phospholipase D with specificity for secondary alcohol transphosphatidylation: Insights into plasticity of substrate binding and activation

2021 ◽  
Author(s):  
Ariela Samantha ◽  
Jasmina Damnjanović ◽  
Yugo Iwasaki ◽  
Hideo Nakano ◽  
Alice Vrielink
Keyword(s):  
Phospholipase D ◽  
Catalytic Mechanism ◽  
Inositol Phosphate ◽  
Substrate Binding ◽  
Secondary Alcohol ◽  
Molecular Size ◽  
Underlying Mechanism ◽  
Head Group ◽  
Wild Type ◽  
Primary Alcohols

Phospholipase D (PLD) is an enzyme useful for the enzymatic modification of phospholipids.  In the presence of primary alcohols, the enzyme catalyses transphosphatidylation of the head group of phospholipid substrates to synthesize a modified phospholipid product.  However, the enzyme is specific for primary alcohols and thus the limitation of the molecular size of the acceptor compounds has restricted the type of phospholipid species that can be synthesised.  An engineered variant of PLD from Streptomycesantibioticus termed TNYR SaPLD was developed capable of synthesizing 1-phosphatidylinositol with positional specificity of up to 98%. To gain a better understanding of the substrate binding features of the TNYR SaPLD, crystal structures have been determined for the free enzyme and its complexes with phosphate, phosphatidic acid and 1-inositol phosphate.  Comparisons of these structures with the wild-type SaPLD show a larger binding site able to accommodate a bulkier secondary alcohol substrate as well as changes to the position of a flexible surface loop proposed to be involved in substrate recognition.  The complex of the active TNYR SaPLD with 1-inositol phosphate reveals a covalent intermediate adduct with the ligand bound to H442 rather than to H168, the proposed nucleophile in the wild type enzyme.  This structural feature suggests that the enzyme exhibits plasticity of the catalytic mechanism different from what has been reported to date for PLDs.  These structural studies provide insights into the underlying mechanism that governs the recognition of myo-inositol by TNYR SaPLD, and an important foundation for further studies of the catalytic mechanism.

scholarly journals Effect of Metronidazole on the Growth of Vaginal Lactobacilliin vitro

2001 ◽  
Vol 9 (1) ◽  
pp. 41-45 ◽  
Author(s):  
Jose A. Simoes ◽  
Alla A. Aroutcheva ◽  
Susan Shott ◽  
Sebastian Faro
Keyword(s):  
Adverse Effect ◽  
Hydrogen Peroxide ◽  
Antimicrobial Agent ◽  
Clinical Laboratory ◽  
National Committee ◽  
Broth Microdilution ◽  
High Concentration ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
The Ideal

Objective:To determine whether metronidazole has an adverse effect on the growth ofLactobacillus.Methods:Hydrogen peroxide- and bacteriocin-producing strains ofLactobacilluswere used as test strains. Concentrations of metronidazole used ranged from 128 to 7000 μg/ml. Susceptibility to metronidazole was conducted by the broth microdilution method recommended by the National Committee for Clinical Laboratory Standards.Results:Growth ofLactobacilluswas partially inhibited at concentrations between 1000 and 4000 μg/ml (p= 0.014). Concentrations ≥ 5000 μg/ml completely inhibited growth ofLactobacillus. Concentrations between 128 and 256 μg/ml stimulated growth ofLactobacillus(p= 0.025 and 0.005, respectively). Concentrations of metronidazole between 64 and 128 μg/ml or ≥ 512 μg/ml did not have an inhibitory or a stimulatory effect on the growth ofLactobacilluscompared to the control.Conclusions:High concentration of metronidazole, i.e. between 1000 and 4000 μg/ml, partially inhibited the growth ofLactobacillus. Concentrations ≥ 5000 μg/ml completely suppressed the growth ofLactobacillus. Concentrations between ≥ 128 and ≤ 256 μg/ml stimulated the growth ofLactobacillus. Further investigation to determine the ideal concentration of metronidazole is needed in order to use the antimicrobial agent effectively in the treatment of bacterial vaginosis.

Preparation of porphyrin modified CO9S8 nanocomposites and application for colorimetric biosensing of H2O2

2018 ◽  
Vol 22 (09n10) ◽  
pp. 935-943 ◽  
Author(s):  
Yan Gao ◽  
Chunqiao Jin ◽  
Miaomiao Chen ◽  
Xixi Zhu ◽  
Min Fu ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Catalytic Activity ◽  
Peroxidase Activity ◽  
Catalytic Mechanism ◽  
Color Change ◽  
Colorimetric Detection ◽  
Buffer Solution ◽  
Hydrogen Peroxide Detection ◽  
Steady State Kinetics ◽  
One Step

Hydrogen peroxide detection has been widely applied in the fields of biology, medicine, and chemistry. Colorimetric detection of hydrogen peroxide has proven to be a fast and convenient method. In this work, 5,10,15,20-tetrakis(4-chlorophenyl) porphyrin modified Co[Formula: see text]S[Formula: see text] nanocomposites (H[Formula: see text]TClPP-Co[Formula: see text]S[Formula: see text] were prepared via a facile one-step hydrothermal method. H[Formula: see text]TClPP-Co[Formula: see text]S[Formula: see text] nanocomposites were demonstrated to possess an enhanced mimetic peroxidase activity toward the substrate, 3,3[Formula: see text],5,5[Formula: see text]-tetramethylbenzidine (TMB), which can be oxidized to oxTMB (oxidized TMB) in a buffer solution of hydrogen peroxide with a color change from colorless to blue. The catalytic activity of H[Formula: see text]TClPP-Co[Formula: see text]S[Formula: see text] was further analyzed by steady-state kinetics, and H[Formula: see text]TClPP-Co[Formula: see text]S[Formula: see text] had high affinity towards both TMB and H[Formula: see text]O[Formula: see text]. Furthermore, fluorescence and ESR data revealed that the catalytic mechanism of the peroxidase activity of H[Formula: see text]TClPP-Co[Formula: see text]S[Formula: see text] is due to hydroxyl radicals generated from decomposition of H[Formula: see text]O[Formula: see text]. Based on the catalytic activity of H[Formula: see text]TClPP-Co[Formula: see text]S[Formula: see text], a sensitive colorimetric sensor of H[Formula: see text]O[Formula: see text] with a detection limit of 6.803 [Formula: see text]M as well as a range of 7–100 [Formula: see text]M was designed.

scholarly journals High-resolution neutron crystallography visualizes an OH-bound resting state of a copper-containing nitrite reductase

2020 ◽  
Vol 117 (8) ◽  
pp. 4071-4077 ◽  
Author(s):  
Yohta Fukuda ◽  
Yu Hirano ◽  
Katsuhiro Kusaka ◽  
Tsuyoshi Inoue ◽  
Taro Tamada
Keyword(s):  
Computational Chemistry ◽  
Structural Biology ◽  
Resting State ◽  
Catalytic Mechanism ◽  
Low Ph ◽  
Intramolecular Electron Transfer ◽  
Hydrogen Atoms ◽  
X Ray ◽  
Electron Transfer Pathway ◽  
Neutron Structure

Copper-containing nitrite reductases (CuNIRs) transform nitrite to gaseous nitric oxide, which is a key process in the global nitrogen cycle. The catalytic mechanism has been extensively studied to ultimately achieve rational control of this important geobiochemical reaction. However, accumulated structural biology data show discrepancies with spectroscopic and computational studies; hence, the reaction mechanism is still controversial. In particular, the details of the proton transfer involved in it are largely unknown. This situation arises from the failure of determining positions of hydrogen atoms and protons, which play essential roles at the catalytic site of CuNIRs, even with atomic resolution X-ray crystallography. Here, we determined the 1.50 Å resolution neutron structure of a CuNIR from Geobacillus thermodenitrificans (trimer molecular mass of ∼106 kDa) in its resting state at low pH. Our neutron structure reveals the protonation states of catalytic residues (deprotonated aspartate and protonated histidine), thus providing insights into the catalytic mechanism. We found that a hydroxide ion can exist as a ligand to the catalytic Cu atom in the resting state even at a low pH. This OH-bound Cu site is unexpected from previously given X-ray structures but consistent with a reaction intermediate suggested by computational chemistry. Furthermore, the hydrogen-deuterium exchange ratio in our neutron structure suggests that the intramolecular electron transfer pathway has a hydrogen-bond jump, which is proposed by quantum chemistry. Our study can seamlessly link the structural biology to the computational chemistry of CuNIRs, boosting our understanding of the enzymes at the atomic and electronic levels.

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