* All experimental methods are cited from the reference, please refer to the original source for details. We do not guarantee the accuracy of the content in the reference.
Reference:
[1] Food and Chemical Toxicology, 1998, vol. 36, # 9-10, p. 761 - 770
[2] Food and Chemical Toxicology, 1999, vol. 37, # 6, p. 655 - 661
[3] Drug Metabolism and Disposition, 2014, vol. 42, # 3, p. 377 - 383
2
[ 56-45-1 ]
[ 72-89-9 ]
[ 5147-00-2 ]
Reference:
[1] Phytochemistry, 2005, vol. 66, # 12, p. 1407 - 1416
[2] Biochimica et Biophysica Acta - Proteins and Proteomics, 2014, vol. 1844, # 10, p. 1741 - 1748
3
[ 56-86-0 ]
[ 72-89-9 ]
[ 1188-37-0 ]
Reference:
[1] Cell Chemical Biology, 2016, vol. 23, # 8, p. 935 - 945
With hydrogenchloride; potassium hydrogencarbonate; In water; acetonitrile; for 0.25h;pH 7.5;Cooling with ice;
General procedure: A solution of <strong>[85-61-0]coenzyme A</strong> (22 mumol) was prepared in 1 mL 0.5 M KHCO3. The pH was brought to approximately 7.5 by addition of 100 muL 1 M HCl, and the solution was placed on ice. To this solution was added an ice-cold solution of succinic or acetic anhydride (35 mumol) in 500 muL acetonitrile. The mixture was incubated on ice for 15 min. Reaction progress was monitored by Ellman assay; if necessary, additional anhydride solution was added. Upon reaction completion, 3 muL of 6 M HCl was added to lower the pH to approximately 6. Acetonitrile was removed in vacuo, and acyl <strong>[85-61-0]coenzyme A</strong> was purified by Supelco LC-18 column with elution in 1% trifluoroacetic acid and 50% acetonitrile. Acetonitrile was removed in vacuo, followed by lyophilization. The product was dissolved in water to 10 mM and stored in aliquots at-80 C.
With serine acetyltransferase from Brucella abortus; In aq. acetate buffer; at 20℃; for 0.0333333h;Enzymatic reaction;Kinetics;
BaSAT activity was calculated by monitoring the decrease in absorbance at 232 nm (A232) due to the breakage of the thioester bond of acetyl coenzyme A using an Ultrospec 2100pro spectrophotometer (GE Healthcare). For this assay, the reaction mixture contained 50 mM Tris-HCl (pH 8.0), 0.1 mM acetyl-CoA, and 2.5 mug of BaSAT. The reaction was started by adding serine, and the absorbance was monitored every 2 s starting from zero. To calculate Km for the binding of serine, the amino acid was added in varying concentrations from 10 to 500 muM, keeping the concentration of acetyl CoA constant at 0.1 mM, and the reaction was carried out at room temperature for 2 min. The Km and the Vmax values were calculated using the Michaelis-Menten curve. Km was similarly calculated for acetyl Co-A, by varying acetyl Co-A from 10 to 400 muM and keeping the concentration of serine constant at 120 muM. Kinetic studies were performed in triplicate using 7-8 different concentrations of both serine and acetyl CoA. To measure the inhibition by l-cysteine, similar reactions were repeated using two different concentrations of cysteine, at 5 and 10 muM. The velocity of the reaction was calculated from the linear part of the time curve (30 s).
With sea bream arylalkylamine N-acetyltransferase containing I157M mutation at β5 region Enzymatic reaction;
With 5,5'-dithiobis-(2-nitrobenzoic acid); Drosophila melanogaster arylalkylamine N-acetyltransferase A from E. coli In aq. buffer Enzymatic reaction;
With N-acyltransferase 23DmAT In water at 25℃; for 18h; Enzymatic reaction;
14.14.2.1 14.2.1 Preliminary assay: Activity towards a panel of amines 36a-d
General procedure: Amine solution (12.5 μΙ) and CoA ester assay mix (37.5 μΙ) were added to each well of a 96-well microtitre plate. Using the Biomek FX liquid handling robot, N-acyltransferase lysate (50 μΙ) was added and reactions were incubated at 25 °C, 800 rpm, for 18 hours.
With dipyridin-4-yl disulfide; wild-type arylalkylamine N-acyltransferase from Tribolium castaneum In aq. buffer Enzymatic reaction;
With recombinant Bacillus cereus arylamine N-acetyltransferase 3 wild-type protein; In aq. buffer;pH 7.5;Enzymatic reaction;Kinetics;
General procedure: Steady-state kinetic experiments were conducted using the 5,5-dithiobis-(nitrobenzoic acid) assay (24) to determine the apparent kinetics of (BACCR)NAT3 and E123D variant enzymes parameters Kmapp and kcat toward 4-aminosalicylic acid, <strong>[153-78-6]2-aminofluorene</strong>, and isoniazid. Initial velocities (Vi) were determined in the presence of arylamine substrate at various concentrations, 400M AcCoA, and 0.5 mug/ml enzyme in 25 mM Tris-HCl, pH 7.5. Alter natively, various concentrations of acetyl donors were used with 500M 4-aminosalicylic acid to determine the apparentkinetic parameters toward AcCoA. The PNPA assaydescribed by Cleland and Hengge (25) was used to obtain kinetic parameters toward the acetyl donor PNPA. Control experiments were conducted in the absence of arylamine substrate. Vi values were plotted against substrate concentration and fitted to the Michaelis-Menten equation using Kaleidagraph 3.5 (Synergy Software). Results of triplicate experiment sare shown including standard deviations.
14
[ 64-17-5 ]
[ 72-89-9 ]
[ 524-14-1 ]
[ 2524-37-0 ]
Yield
Reaction Conditions
Operation in experiment
With Armillaria mellea iterative type I polyketide synthase ArmB, N-terminal hexahistidine-tagged protein; In aq. phosphate buffer; for 20h;pH 7.4;Enzymatic reaction;
General procedure: Approximately 20mug ArmB was incubated with acetyl-CoA (1mM) and malonyl-CoA (3mM) in 100mul reaction buffer (100mM NaH2PO4, pH= 7.4). As a negative control, we used a protein fraction obtained from E.coli BL21 transformed with the empty vector pET28a instead of the armB expression plasmid pGL77. For the formation of esters, 20mM alcohol was added from200mM stock solutions. Relative yields of alcohols were calculated from areas under curve (AUCs; lambda= 254nm) of OA derivatives as follows: AUCEster/(AUCEster+ AUCOA). For melleolide assembly invitro, the sesquiterpene alcohol was directly dissolved in reaction buffer to obtain an approximate 5mM solution. After 20hr, the reaction mixture was extracted twice with one volume of ethyl acetate/acetic acid (99:1), evaporated to dryness, and solved in 100mul MeOH for high-performance liquid chromatography (HPLC) and MS measurements. Analytical HPLC was performed on an Agilent 1200 instrument equipped with a diode array detector, set to detect the wavelength range from 210 to 400nm, and a Zorbax Eclipse XDB-C18 column (150× 4.6mm). HPLC conditions were as follows: gradient elution with 0.1% trifluoroacetate in H2O/MeCN 99:1 to 100% MeCN in 30min, MeCN 100% for 12min, and flow rate 1ml/min. High-resolution electrospray ionization-MS was measured on a Thermo Accela instrument (LC) and a Thermo Exactive orbitrap spectrometer (high-resolution MS) in negative mode.
With Armillaria mellea iterative type I polyketide synthase ArmB, N-terminal hexahistidine-tagged protein In aq. phosphate buffer for 20h; Enzymatic reaction;
Polyketide Synthase In Vitro Reaction and Product Analysis
General procedure: Approximately 20μg ArmB was incubated with acetyl-CoA (1mM) and malonyl-CoA (3mM) in 100μl reaction buffer (100mM NaH2PO4, pH= 7.4). As a negative control, we used a protein fraction obtained from E.coli BL21 transformed with the empty vector pET28a instead of the armB expression plasmid pGL77. For the formation of esters, 20mM alcohol was added from200mM stock solutions. Relative yields of alcohols were calculated from areas under curve (AUCs; λ= 254nm) of OA derivatives as follows: AUCEster/(AUCEster+ AUCOA). For melleolide assembly invitro, the sesquiterpene alcohol was directly dissolved in reaction buffer to obtain an approximate 5mM solution. After 20hr, the reaction mixture was extracted twice with one volume of ethyl acetate/acetic acid (99:1), evaporated to dryness, and solved in 100μl MeOH for high-performance liquid chromatography (HPLC) and MS measurements. Analytical HPLC was performed on an Agilent 1200 instrument equipped with a diode array detector, set to detect the wavelength range from 210 to 400nm, and a Zorbax Eclipse XDB-C18 column (150× 4.6mm). HPLC conditions were as follows: gradient elution with 0.1% trifluoroacetate in H2O/MeCN 99:1 to 100% MeCN in 30min, MeCN 100% for 12min, and flow rate 1ml/min. High-resolution electrospray ionization-MS was measured on a Thermo Accela instrument (LC) and a Thermo Exactive orbitrap spectrometer (high-resolution MS) in negative mode.
With 5,5'-dithiobis-(2-nitrobenzoic acid); Drosophila melanogaster Arylalkylamine N?Acetyltransferase Like 7 expressed in Escherichia coli; In aq. buffer; at 22℃; for 0.5h;pH 8.0;Enzymatic reaction;Kinetics;
General procedure: Ellman?s reagent20was used to assay the AANATL7 activity by measuring therelease of coenzyme A at 412 nm with a molar extinctioncoefficient of 13600 M-1 cm-1 in a buffered solution containing300 mM Tris (pH 8.0), 150 muM DTNB, and the desiredconcentrations of substrates and/or inhibitors. Initial velocitieswere measured by using a Cary 300 Bio UV-visiblespectrophotometer at 22 C. The apparent kinetic constantswere generated for each amine substrate by holding acetyl-CoAat a fixed saturating concentration. Apparent kinetic constantsfor each acyl-CoA substrate were determined by holdinghistamine at a fixed saturating concentration. The resulting datawere fit to eq 1 using Sigma Plot 12.0 to generate the steadystatekinetic constants=+vVK[S][S] omax,appm,app (1)where vo is the initial velocity, Vmax,app is the apparent maximalvelocity, Km,app is the apparent Michaelis constant, and [S] isthe substrate concentration. Each assay was performed intriplicate. Uncertainty calculations for kcat,app, (kcat/Km)app, andrelative (kcat/Km)app were calculated using eq 2sigmasigma sigma= + xyxy x yx y2 2(2)where sigma is the standard error.21
With glucosamine N-acetyltransferase from clostridium acetobutylicum; In aq. buffer; at 37℃; for 2h;pH 7;Enzymatic reaction;
General procedure: The acceptor specificity of GlmA, RmNag and Naa10p toward hexoamineswas explored by using GlcN, GalN and ManN. The reactionswere carried out in a final volume of 30 muL containing 100mM Tirs-HCl(pH 7.0 for GlmA; pH 7.5 for RmNag; and pH 8.0 for Naa10p), 5mMvaried hexoamines (GlcN, GalN or ManN), 1.5mM Acetyl-CoA and10 muM different enzymes (GlmA, RmNag or Naa10p). The reactionmixtures were incubated at 37 C for 2 h, and then 1 muL portions of thereaction mixtures were applied to thin-layer chromatography (TLC,silica gel F254, Merck) analysis. The reaction mixtures were developedby n-butanol/methanol/ammonia/water=5:4:2:1 (V/V/V/V) andstained with p-anisaldehyde solution (ethanol/sulfuric acid/p-anisaldehyde/acetic acid=500:27:16:5.5). Reaction mixtures withoutacceptor substrate were used as controls.
With triethanolamine; ethylenediaminetetraacetic acid; carnitine acetyltransferase; arylamine N-acetyltransferase 2*4, wild-type allele; acetylcarnitine; diothiothreitol In water at 37℃; for 18h; Enzymatic reaction;
With triethanolamine; ethylenediaminetetraacetic acid; carnitine acetyltransferase; arylamine N-acetyltransferase 2*4, wild-type allele; acetylcarnitine; diothiothreitol In water at 37℃; for 18h; Enzymatic reaction;
With triethanolamine; ethylenediaminetetraacetic acid; carnitine acetyltransferase; arylamine N-acetyltransferase 2*4, wild-type allele; acetylcarnitine; diothiothreitol In water at 37℃; for 18h; Enzymatic reaction;
With whole-cell containing E. coli PABH with expressed 4-aminobenzoate hydroxylaseABH60G272V/T290V; whole-cell containing E. coli PAAT with expressed arylamine N-acetyltransferasePANATK211G at 30℃; for 7h; Green chemistry; Enzymatic reaction;