scholarly journals Functional analysis of (4S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase

2015 ◽  
Vol 112 (11) ◽  
pp. 3332-3337 ◽  
Author(s):  
Narayanan Srividya ◽  
Edward M. Davis ◽  
Rodney B. Croteau ◽  
B. Markus Lange
Keyword(s):  
Enzyme Activity ◽  
Critical Role ◽  
Enzymatic Reaction ◽  
Site Mutation ◽  
Alanine Scanning Mutagenesis ◽  
Sequence Comparisons ◽  
Significant Drop ◽  
Mentha Spicata ◽  
Aromatic Residues ◽  
Limonene Synthase

Crystal structural data for (4S)-limonene synthase [(4S)-LS] of spearmint (Mentha spicata L.) were used to infer which amino acid residues are in close proximity to the substrate and carbocation intermediates of the enzymatic reaction. Alanine-scanning mutagenesis of 48 amino acids combined with enzyme fidelity analysis [percentage of (−)-limonene produced] indicated which residues are most likely to constitute the active site. Mutation of residues W324 and H579 caused a significant drop in enzyme activity and formation of products (myrcene, linalool, and terpineol) characteristic of a premature termination of the reaction. A double mutant (W324A/H579A) had no detectable enzyme activity, indicating that either substrate binding or the terminating reaction was impaired. Exchanges to other aromatic residues (W324H, W324F, W324Y, H579F, H579Y, and H579W) resulted in enzyme catalysts with significantly reduced activity. Sequence comparisons across the angiosperm lineage provided evidence that W324 is a conserved residue, whereas the position equivalent to H579 is occupied by aromatic residues (H, F, or Y). These results are consistent with a critical role of W324 and H579 in the stabilization of carbocation intermediates. The potential of these residues to serve as the catalytic base facilitating the terminal deprotonation reaction is discussed.

The Role of Glyoxalase in Glycation and Carbonyl Stress Induced Metabolic Disorders

2020 ◽  
Vol 21 (9) ◽  
pp. 846-859
Author(s):  
Mohd Saeed ◽  
Mohd Adnan Kausar ◽  
Rajeev Singh ◽  
Arif J. Siddiqui ◽  
Asma Akhter
Keyword(s):  
Protein Misfolding ◽  
Covalent Binding ◽  
Critical Role ◽  
Enzymatic Reaction ◽  
Treatment Strategies ◽  
Bioactive Molecules ◽  
Carbonyl Stress ◽  
Defense System ◽  
Pathological Conditions ◽  
Age Related

Glycation refers to the covalent binding of sugar molecules to macromolecules, such as DNA, proteins, and lipids in a non-enzymatic reaction, resulting in the formation of irreversibly bound products known as advanced glycation end products (AGEs). AGEs are synthesized in high amounts both in pathological conditions, such as diabetes and under physiological conditions resulting in aging. The body’s anti-glycation defense mechanisms play a critical role in removing glycated products. However, if this defense system fails, AGEs start accumulating, which results in pathological conditions. Studies have been shown that increased accumulation of AGEs acts as key mediators in multiple diseases, such as diabetes, obesity, arthritis, cancer, atherosclerosis, decreased skin elasticity, male erectile dysfunction, pulmonary fibrosis, aging, and Alzheimer’s disease. Furthermore, glycation of nucleotides, proteins, and phospholipids by α-oxoaldehyde metabolites, such as glyoxal (GO) and methylglyoxal (MGO), causes potential damage to the genome, proteome, and lipidome. Glyoxalase-1 (GLO-1) acts as a part of the anti-glycation defense system by carrying out detoxification of GO and MGO. It has been demonstrated that GLO-1 protects dicarbonyl modifications of the proteome and lipidome, thereby impeding the cell signaling and affecting age-related diseases. Its relationship with detoxification and anti-glycation defense is well established. Glycation of proteins by MGO and GO results in protein misfolding, thereby affecting their structure and function. These findings provide evidence for the rationale that the functional modulation of the GLO pathway could be used as a potential therapeutic target. In the present review, we summarized the newly emerged literature on the GLO pathway, including enzymes regulating the process. In addition, we described small bioactive molecules with the potential to modulate the GLO pathway, thereby providing a basis for the development of new treatment strategies against age-related complications.

Enzymatic condensation of dopamine and acetaldehyde: a salsolinol synthase from rat brain

Biologia ◽  
2011 ◽  
Vol 66 (6) ◽  
Author(s):  
Xuechai Chen ◽  
Abida Arshad ◽  
Hong Qing ◽  
Rui Wang ◽  
Jianqing Lu ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Substantia Nigra ◽  
Human Subjects ◽  
Enzymatic Reaction ◽  
Brain Regions ◽  
Activity Detection ◽  
Reaction Products ◽  
Sprague Dawley ◽  
Sprague Dawley Rats ◽  
Salsolinol Synthase

AbstractSalsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; Sal) is structurally similar to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, which is supposed to have a role in the development of Parkinson-like syndrome in both human and non-human subjects. In the human brain, the amount of (R)-enantiomer of Sal is much higher than (S)-enantiomer, suggesting that a putative enzyme may participate in the synthesis of (R)-salsolinol, called (R)-salsolinol synthase. In this study, the (R)-salsolinol synthase activity in the condensation of dopamine and acetaldehyde was investigated in the crude extracts from the brains of Sprague Dawley rats. Identification of the enzymatic reaction products and enzyme activity detection were achieved by HPLC-electrochemical detection. The discovery of this enzyme activity in rat’s brain indicates the natural existence of (R)-salsolinol synthase in the brains of humans and rats, and it is distributed in most brain regions of rat with higher activity in soluble proteins extracted from striatum and substantia nigra.

scholarly journals Pre-fermentative treatment of a model wine with aim to serve as a functional food with decreased alcohol content

2017 ◽  
Vol 63 (01) ◽  
pp. 47-53
Author(s):  
Irina Mladenoska ◽  
Verica Petkova ◽  
Tatjana Kadifkova Panovska
Keyword(s):  
Enzyme Activity ◽  
Gluconic Acid ◽  
Functional Food ◽  
Ph Value ◽  
Enzymatic Reaction ◽  
Alginate Beads ◽  
High Concentration ◽  
Ph Optimum ◽  
Enzyme Preparations ◽  
Initial Ph

The effect of substrate concentration on the enzyme activity in the reaction of glucose conversion into gluconic acid was investigated by using three different enzyme preparations in media with two different glucose concentrations. The media were simulating the conditions in the must, thus named as minimal model must, and were composed form combination of several organic acids and glucose. Those media were having initial pH of 3.5 that is a very unfavorable for glucose oxidase activity having a pH optimum at the pH value of 5.5. Among the three preparations used, the bakery additive, Alphamalt Gloxy 5080, was the most active in the medium with glucose concentration of 10 g/L, showing conversion of more than 70% for the period of 24 h, while the same enzyme preparation in the medium with 100 g/L glucose converted only about 7% of glucose. The pH value of the medium at the beginning and at the end of the enzymatic reaction was a good indicator of the enzyme activity. It seems that for the conversion of glucose in higher concentration, enzymatic preparation in high concentration should also be used. The preliminary attempt of immobilization of two preparations of glucose oxidases in alginate beads was also performed and a successful immobilization procedure for utilization in food industry was preliminarily developed. Keywords: glucose oxidases, enzymatic pretreatment, glucose, gluconic acid, model wine, functional food

scholarly journals Evaluation of the Effect of Irrigation on Biometric Growth, Physiological Response, and Essential Oil of Mentha spicata (L.)

Water ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 2264 ◽  
Author(s):  
Marino ◽  
Ahmad ◽  
Ferreira ◽  
Alvino
Keyword(s):  
Water Stress ◽  
Essential Oil ◽  
Water Status ◽  
Stomatal Closure ◽  
Critical Role ◽  
Net Photosynthesis ◽  
Leaf Biomass ◽  
Water Losses ◽  
Mentha Spicata ◽  
Area Index

A field experiment was performed on spearmint (Mentha spicata L.) under different irrigation regimes in a hilly area of Southern Italy. Objectives of the study include evaluating the physiological and biometrical response of mint from plant establishment up to its complete maturation, as well as the yield composition in essential oil at two different dates. Increasing levels of water stress affected later developing leaves and plant’s water status and net photosynthesis (from the beginning of stress (DAT 63), while affecting negatively the biometric response very soon and significantly from 35 DAT. Photosynthesis limitation played a critical role from DAT 53 on, namely later, in the harvest period (DAT 35–70). Under severe water stress, crop restricted water losses by modulating stomatal closure and, at harvest, showing lowered mesophyll conductance. Irrigation treatments did not affect the concentration of organic compounds, while the yield of essential oils was negatively affected by water stress due to reduced crop growth, in terms of total and leaf biomass, leaf area index (LAI) and crop height.

scholarly journals Effects of music playing on biological molecules

2018 ◽  
Vol 210 ◽  
pp. 05006 ◽  
Author(s):  
Catia Algieri ◽  
Claudio Guarnaccia ◽  
Vincenzo Barone ◽  
Maria Raffaella Gullo ◽  
Laura Donato
Keyword(s):  
Enzyme Activity ◽  
Detrimental Effect ◽  
Specific Activity ◽  
Time Lag ◽  
Enzymatic Reaction ◽  
Biological Molecules ◽  
National Council ◽  
Musical Piece ◽  
Tyrosinase Enzyme ◽  
Positive Effect

In this work the effect of two different musical pieces (called “Reminiscenza” and “Pioggia”) on the tyrosinase enzyme activity was investigated. The l-DOPA production by the enzymatic reaction was measured during the continuous playing of these musical pieces. Experiments were performed in the laboratories of the Institute of Membrane Technology (ITM) of the Italian National Council for Research (CNR). The results showed that a positive effect was exercised by the “Reminiscenza” piece, which determined an increase of the specific activity about of 30 % with respect to the value measured in the absence of sound. On the contrary, “Pioggia” piece had a detrimental effect on the enzymatic process. In particular, a time lag on the l-DOPA production, during the first minutes of the reaction, was detected. After this period, an increase of the reaction velocity occurred even if the enzyme activity was lower than the value obtained in the absence of music. These results show that the peculiar characteristics of a musical piece can exercise a positive or negative action on biological elements and open the way to further studies in this area.

Fluorometric Method for Measuring Serum Lipase Activity

1975 ◽  
Vol 21 (12) ◽  
pp. 1788-1790 ◽  
Author(s):  
Bernd Rietz ◽  
George G Guilbault
Keyword(s):  
Enzyme Activity ◽  
Olive Oil ◽  
Lipase Activity ◽  
Enzymatic Reaction ◽  
Fluorometric Method ◽  
Reaction Volume ◽  
Serum Lipase ◽  
Fluorescent Indicator ◽  
Substrate Solution ◽  
Emulsified Olive Oil

Abstract We describe a method for determining lipase (triacylglycerol acyl-hydrolase, EC 3.1.1.3) activity in serum. Emulsified olive oil in tris(hydroxymethyl)aminomethane buffer is used as substrate. The course of the enzymatic reaction is followed by measuring the decrease in fluorescence with time of a fluorescent indicator, 4-methylumbelliferone, added to the substrate, caused by the change in the pH of the substrate solution resulting from the reaction. All measurements are performed at 37 °C. The reaction volume is about 2.3 ml, in a Pyrex cuvette. Change of fluorescence and of enzyme activity are linearly related in the range of 77.8 to 389.0 U/liter. An assay can be done in as little as 3 to 5 min, with excellent precision.

Studies on Partially Purified Pig Liver Steroid Δ4-5β-Reductase Activity

1973 ◽  
Vol 51 (12) ◽  
pp. 1661-1668 ◽  
Author(s):  
Edward J. Van Doorn ◽  
John C. Nduaguba ◽  
Albert F. Clark
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Enzyme Preparation ◽  
Reductase Activity ◽  
Enzymatic Reaction ◽  
Ph Optimum ◽  
Pig Liver ◽  
Liver Cytosol ◽  
End Products ◽  
Tris Maleate

Some properties of partially purified steroid Δ4-5β-reductase activity of pig liver cytosol have been studied using testosterone as substrate. The enzymatic activity was stable for 72 h at 4° when stored in 0.05 M Tris–maleate buffer, pH 7.4 or 8.4; storage at pH 8 at 4° resulted in a 25% decrease in activity in 30 days. The pH optimum in Tris–maleate buffers was 6.4. Enzyme activity was completely inhibited by 0.2 mM p-chloromercuribenzenesulfonate and 0.2 mM p-chloromercuribenzoate. Enzyme activity was reduced by 20% and 45% with 1.0 mM iodoacetamide and 1.0 mM N-ethylmaleimide, respectively. The end products of the enzymatic reaction, NADP+ and 5β-dihydrotestosterone, inhibited the rate of reduction of testosterone. Testosterone Δ4-5β-reductase activity was present in protein of molecular weight 25 000–30 000, as determined by gel filtration.The enzyme preparation reduced a variety of C19 and C21 steroids. The highest activity (twice that for testosterone) was found with aldosterone as substrate.

scholarly journals Expression, crystallization and structure elucidation of γ-terpinene synthase fromThymus vulgaris

2016 ◽  
Vol 72 (1) ◽  
pp. 16-23 ◽  
Author(s):  
Kristin Rudolph ◽  
Christoph Parthier ◽  
Claudia Egerer-Sieber ◽  
Daniel Geiger ◽  
Yves A. Muller ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Structure Elucidation ◽  
Structural Similarity ◽  
Enzymatic Conversion ◽  
Terpenoid Biosynthesis ◽  
Mentha Spicata ◽  
Geranyl Diphosphate ◽  
Limonene Synthase ◽  
Putative Active Site

The biosynthesis of γ-terpinene, a precursor of the phenolic isomers thymol and carvacrol found in the essential oil fromThymussp., is attributed to the activitiy of γ-terpinene synthase (TPS). Purified γ-terpinene synthase fromT. vulgaris(TvTPS), theThymusspecies that is the most widely spread and of the greatest economical importance, is able to catalyze the enzymatic conversion of geranyl diphosphate (GPP) to γ-terpinene. The crystal structure of recombinantly expressed and purifiedTvTPS is reported at 1.65 Å resolution, confirming the dimeric structure of the enzyme. The putative active site ofTvTPS is deduced from its pronounced structural similarity to enzymes from other species of the Lamiaceae family involved in terpenoid biosynthesis: to (+)-bornyl diphosphate synthase and 1,8-cineole synthase fromSalviasp. and to (4S)-limonene synthase fromMentha spicata.

scholarly journals Production and characterization of staphylokinase enzyme from Staphylococcus aureus ASIA4

2020 ◽  
pp. 027-035
Author(s):  
Alzahrani Nourah Hassan ◽  
El-Shenawy Fareed Shawky
Keyword(s):  
Staphylococcus Aureus ◽  
Enzyme Activity ◽  
Enzymatic Reaction ◽  
Skim Milk ◽  
Casein Hydrolysate ◽  
Fermentation Medium ◽  
University Hospital ◽  
Clinical Samples ◽  
Different Diameters

Six clinical Staphylococcus aureus strains isolated from different clinical samples. Isolates ASIA1 and ASIA2 isolated from urine samples of urinary tract infected patients; ASIA3 isolated from swab samples of burn abscess patients at Assiut University hospital as well as ASIA4, ASIA5 and ASIA6 obtained from blood samples of different cancer patients at South Egypt Cancer Institute. All isolates showed varied abilities to produce halo zones of hydrolysis with different diameters on blood agar, heated plasma agar, casein agar and skim milk agar plates along with different clot lyses percent. Staphylococcus aureus ASIA3, ASIA4 and ASIA6 produced 4.83, 5.98 and 2.08 U/mL of staphylokinase on tryptone soy broth reduced to 1.95, 2.08 and 1.70 U/mL on casein hydrolysate yeast extract broth, respectively. On the other hand, Staphylococcus aureus ASIA1, ASIA2 and ASIA5 gave 2.20, 2.93 and 3.65 U/mL on CYEB compared to 2.10, 1.88 and 3.41 U/mL on TSB as production medium. The staphylokinase yielded from the hyperactive producer Staphylococcus aureus ASIA4 was increased for 7.64-fold (from 2.08 U/mL to 15.88 U/mL) on the optimized fermentation medium composed of 5.0 g sucrose as carbon source, 10.0 g soy bean as nitrogen source, 5.0 g NaCl, K2HPO4 5.0 g and pH 7.0 that inoculated with isolate ASIA4 and incubated for 24 h at 35 °C. Moreover, Staphylokinase activity reached its peak at the optimal enzymatic reaction conditions which were reaction time 25 min, casein as substrate, reaction pH 8.0, reaction temperature 40 °C. In addition it retained 100% of its activity at temperature ranged between 15 and 45 °C and pH ranged from pH 6.0 to 9.0. EDTA inhibited the enzyme activity by 3.0% to 32.2% with increasing its values from 30.0 to 90.0 mM. MgCl2 at a concentration of 30 mM increased the enzyme activity by 4% and then slightly decreased at higher concentrations but NaCl was potent staphylokinase activator at concentrations lower than 90 mM.

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