- Fluorometric coupled enzyme assay for N-sulfotransferase activity of N-deacetylase/N-sulfotransferase (NDST)
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N-Deacetylase/N-sulfotransferases (NDSTs) are critical enzymes in heparan sulfate (HS) biosynthesis. Radioactive labeling assays are the preferred methods to determine the N-sulfotransferase activity of NDST. In this study, we developed a fluorometric coupled enzyme assay that is suitable for the study of enzyme kinetics and inhibitory properties of drug candidates derived from a large-scale in silico screening targeting the sulfotransferase moiety of NDST1. The assay measures recombinant mouse NDST1 (mNDST1) sulfotransferase activity by employing its natural substrate adenosine 3′-phophoadenosine-5′-phosphosulfate (PAPS), a bacterial analog of desulphated human HS, Escherichia coli K5 capsular polysaccharide (K5), the fluorogenic substrate 4-methylumbelliferylsulfate and a double mutant of rat phenol sulfotransferase SULT1A1 K56ER68G. Enzyme kinetic analysis of mNDST1 performed with the coupled assay under steady state conditions at pH 6.8 and 37°C revealed Km (K5) 34.8 μM, Km (PAPS) 10.7 μM, Vmax (K5) 0.53 ± 0.13 nmol/min/μg enzyme, Vmax (PAPS) 0.69 ± 0.05 nmol/min/μg enzyme and the specific enzyme activity of 394 pmol/min/μg enzyme. The pH optimum of mNDST1 is pH 8.2. Our data indicate that mNDST1 is specific for K5 substrate. Finally, we showed that the mNDST1 coupled assay can be utilized to assess potential enzyme inhibitors for drug development.
- Atienza, Joshua,Tkachyova, Ilona,Tropak, Michael,Fan, Xiaolian,Schulze, Andreas
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p. 1093 - 1101
(2021/10/19)
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- Chemo-Enzymatic Synthesis of Position-Specifically Modified RNA for Biophysical Studies including Light Control and NMR Spectroscopy
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The investigation of non-coding RNAs requires RNAs containing modifications at every possible position within the oligonucleotide. Here, we present the chemo-enzymatic RNA synthesis containing photoactivatable or 13C,15N-labelled nucleosides. All four ribonucleotides containing ortho-nitrophenylethyl (NPE) photocages, photoswitchable azobenzene C-nucleotides and 13C,15N-labelled nucleotides were incorporated position-specifically in high yields. We applied this approach for the synthesis of light-inducible 2′dG-sensing riboswitch variants and detected ligand-induced structural reorganization upon irradiation by NMR spectroscopy. This chemo-enzymatic method opens the possibility to incorporate a wide range of modifications at any desired position of RNAs of any lengths beyond the limits of solid-phase synthesis.
- Keyhani, Sara,Goldau, Thomas,Blümler, Anja,Heckel, Alexander,Schwalbe, Harald
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supporting information
p. 12017 - 12021
(2018/09/06)
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- A nano switch mechanism for the redox-responsive sulfotransferase
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Cellular redox signaling is important in diverse physiological and pathological processes. The activity of rat phenol sulfotransferase (rSULT1A1), which is important for the metabolism of hormone and drug, is subjected to redox regulation. Two cysteines, Cys232 and Cys66, nanometer away from each other and from the enzyme active site were proposed to form disulfide bond to regulate the activity of rSULT1A1. A nano switch, composed of a flexible loop from amino acid residues 59-70, explained how this long distance interaction between two cysteines can be achieved. The enzyme properties were investigated through site-directed muatagnesis, circular dichroism, enzyme kinetics and homologous modeling of the rSULT1A1 structures. We proposed that the formation of disulfide bond between Cys232 and Cys66 induced conformational changes of sulfotransferase, then in turn affected its nucleotide binding and enzyme activity. This discovery was extended to understand the possible redox regulation of other sulfotransferases from different organisms. The redox switch can be created in other redox-insensitive sulfotransferases, such as human phenol sulfotransferase (hSULT1A1) and human alcohol sulfotransferase (hSULT2A1), to produce mutant enzymes with redox regulation capacity. This study strongly suggested that redox regulation of drug and hormone metabolism can be significantly varied even though the sequence and structure of SULT1A1 of human and rat have a high degree of homology.
- Lin, Chih-Heng,Lin, En-Shyh,Su, Tian-Mu,Hung, Kuo-Sheng,Yang, Yuh-Shyong
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experimental part
p. 224 - 231
(2012/09/07)
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- Structure-activity relationships for hydroxylated polychlorinated biphenyls as inhibitors of the sulfation of dehydroepiandrosterone catalyzed by human hydroxysteroid sulfotransferase SULT2A1
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Polychlorinated biphenyls (PCBs) are persistent worldwide pollutants that are of concern due to their bioaccumulation and health effects. Metabolic oxidation of PCBs results in the formation of hydroxylated metabolites (OHPCBs). Among their biological effects, OHPCBs have been shown to alter the metabolism of endocrine hormones, including inhibition of mammalian cytosolic sulfotransferases (SULTs) that are responsible for the inactivation of thyroid hormones and phenolic steroids (i.e., hSULT1A1, hSULT1B1, and hSULT1E1). OHPCBs also interact with a human hydroxysteroid sulfotransferase that plays a role in the sulfation of endogenous alcohol-containing steroid hormones and bile acids (i.e., hSULT2A1). The objectives of our current study were to examine the effects of a series of OHPCB congeners on the activity of hSULT2A1 and to develop a three-dimensional quantitative structure-activity relationship (3D-QSAR) model for OHPCBs as inhibitors of the enzyme. A total of 15 OHPCBs were examined, and the sulfation of 1 μM [3H] dehydroepiandrosterone (DHEA) was utilized as a model reaction catalyzed by the enzyme. All 15 OHPCBs inhibited the sulfation of DHEA, with IC50 values ranging from 0.6 μM to 96 μM, and eight of these OHPCBs were also substrates for the enzyme. Comparative molecular field analysis (CoMFA) provided a predictive 3D-QSAR model with a q2 value of 0.697 and an r 2 value of 0.949. The OHPCBs that had the highest potency as inhibitors of DHEA sulfation were those with a 3, 5-dichloro-4-hydroxy substitution pattern on the biphenyl ring system, and these congeners were also substrates for sulfation catalyzed by hSULT2A1.
- Ekuase, Edugie J.,Liu, Yungang,Lehmler, Hans-Joachim,Robertson, Larry W.,Duffel, Michael W.
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experimental part
p. 1720 - 1728
(2012/05/04)
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- An improved one-pot synthesis of nucleoside 5'-triphosphate analogues
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Nucleoside 5'-triphosphate (NTP) analogues are valuable tools for biochemical and medicinal research. Therefore, a facile and efficient synthesis of NTP analogues is required. Here, we report on an improved nucleoside 5'-triphosphorylation procedure to obtain pure products after liquid chromotagrpahy (LC) separation with no need for high performance liquid chromatography (HPLC) purification. To improve the selectivity of the reaction we attempted the optimization of several parameters such as solvent, pyrophosphate nucleophilicity, time and temperature of the reaction. Eventually, the reaction was optimized by decreasing the temperature to -15°C and increasing the reaction time to 2 hours, based on monitoring time-dependent product distribution using 31P NMR. Furthermore, the NTPs were obtained as pure products after LC separation, which was impossible in the original Ludwig procedure. Good yields were obtained for all studied natural and synthetic nucleosides.
- Gillerman, Irina,Fischer, Bilha
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scheme or table
p. 245 - 256
(2011/08/06)
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- Phosphorylation of Nucleotides with Inorganic Cyclo-Triphosphate
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Phosphorylation of nucleotides (nucleoside 3'- and 5'-monophosphates, and 2'-deoxynucleoside 5'-monophosphates) with inorganic sodium cyclotriphosphate (P3m) was studied in aqueous solutions under various conditions (mixing ratio of P3m to nucleotides, pH, reaction temperature, and time). (1) Unprotected nucleoside 5'-monophosphates (5'-NMP's) were easily phosphorylated at the cis-2',3'-diol by P3m to form selectively nucleoside 2',5'-bis(monophosphate) (2',5'-NDP's), nucleoside 3',5'-bis(monophosphate) (3',5'-NDP's), and nucleoside 2',3'-cyclic 5'-bis(monophosphate) (cNDP's). (2) The phosphorylation of 5'-NMP's was strongly dependent on mixing ratio, pH, reaction temperature, and time.Under conditions of high mixing ratios of P3m to 5'-NMP's (5:1 - 10:1), high pH (12), and room temperature, 92 -98percent of 5'-NMp's was converted into 3',5'-NDP's and 2',5'-NDP's in roughly equimolar quantities. (3) Small quantities (5 -8percent) of cNdP's were formed at the initial stage of reaction of 5'-NMP's with P3m but in the course of the reaction for a long period, cNDP's were hydrolyzed to 2',5'-NDP's and 3',5'-NDP's. (4) Nucleoside 3'-monophosphates (3'-NMP's) and 2'-deoxynucleoside 5'-monophosphates (dNMP's) could not be phosphorylated by P3m, which indicates that the presence of hydroxyl groups at both 2'- and 3'-positions on nucleotides is indispensable for the phosphorylation of nucleotides with P3m. (5) The mechanism of the formation of 2',5'-NDP's, 3',5'-NDP's, and cNDP's in the phosphorylation of 5'-NMP's with P3m is discussed.
- Tsuhako, Mitsutomo,Kunitomi, Rumi,Baba, Yoshinobu,Miyajima, Tohru
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p. 490 - 497
(2007/10/02)
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