Fluorometric coupled enzyme assay for N-sulfotransferase activity of N-deacetylase/N-sulfotransferase (NDST)

Abstract

N-Deacetylase/N-sulfotransferases (NDST) 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.

Structural and mechanistic characterization of bifunctional heparan sulfate N-deacetylase-N-sulfotransferase 1

“…We tested the in vitro enzymatic activity of solubilized NDST1 using a recently described coupled enzyme system, in which NDST1 activity is linked to the rat sulfotransferase Sult1A1, which catalytically regenerates sulfate donor PAPS from fluorogenic 4-methylumbelliferyl (4-MU) sulfate 34 (Supplementary Fig. 2a, b ).…”

Structural and mechanistic characterization of bifunctional heparan sulfate N-deacetylase-N-sulfotransferase 1

“…We tested the in vitro enzymatic activity of solubilized NDST1 using a recently described coupled enzyme system, in which NDST1 activity is linked to the rat sulfotransferase Sult1A1, which catalytically regenerates sulfate donor PAPS from fluorogenic 4-methylumbelliferyl (4-MU) sulfate 32 ( Figure S2a, b ). We carried out initial activity trials using Mg 2+ supplemented in the assay buffer, to satisfy the requirement of NDST1 catalysis on divalent cations, which are necessary for deacetylation.…”

Synthesis of bioengineered heparin by recombinant yeast Pichia pastoris

“…We firstly attempted to express the N-terminal signal peptide (43 amino acids, targeting the Golgi apparatus) deleted mutants of hNDST (human) and mNDST (mouse) in E. coli BL21(DE3), S. cerevisiae and P. pastoris , and assayed the activities of N -deacetylase/ N -sulfotransferase within cell lysates. In view of the sequential deacetylation and sulfation reactions, and inspired by the developed fluorometric coupled enzyme assay for NDST, 34 the two domains N -deacetylase and N -sulfotransferase of NDST were coupled for analysis to evaluate its overall catalytic efficiency. Compared with mNDST ΔN43 , hNDST ΔN43 showed higher N -deacetylase/ N -sulfotransferase activity in both S. cerevisiae (28.8 U L −1 ) and P. pastoris (94.9 U L −1 ) lysates (Fig.…”

“…N -Deacetylase/ N -sulfotransferase activity 34 was determined in 1.5 mL reaction solution containing 50 mM p -nitrophenyl sulfate (PNPS), 0.5 mM 3′-phosphoadenosine-5′-phosphate (PAP), 2 mg aryl sulfotransferase IV (AST IV), 35 20% (v/v) glycerol, 2 mg mL −1 heparosan and 500 μL different NDST mutant cell lysates. The reaction system was incubated at 40 °C for 2 h.…”