Activity assays using the Malachite Green Assay

The amount of free phosphate during the dephosphorylation of phosphosubstrates was determined with the malachite green assay (Sigma-Aldrich) according to the manufacturer’s protocol. The substrates tested were: phosphoinositide substrates diC8-PI3P, diC8-PI(3,4)P₂, diC8-PI(3,5)P₂, diC8-PI4P, diC8-PI(4,5)P₂, diC8-PI5P, and diC8-PI(3,4,5)P₃ (all from Echelon Bioscience), O-phospho-L-tyrosine (P-Tyr), O-phospho-L-serine (P-Ser), O-phospho-L-threonine (P-Thr), cysteamine S-phosphate sodium salt (CysS-P), bis-(p-nitrophenyl) phosphate sodium salt (Bis pNPP), phosphorylcholine chloride (PC), O-phosphorylethanolamine (PETh), mono-sodium phosphoenolpyruvate hydrate (PEP), glycerophosphate disodium salt hydrate (GlyP), inosine 5′-monophosphate disodium salt hydrate (IMP), guanosine 5′-monophosphate disodium salt hydrate (GMP), guanosine 5′-triphosphate sodium salt hydrate (GTP), adenosine 5′-triphosphate (ATP) disodium salt hydrate, adenosine 5′-diphosphate sodium salt (ADP), adenosine 5′-monophosphate disodium salt (AMP), trehalose 6-phosphate dipotassium salt (T6P), D-fructose 6-phosphate disodium salt hydrate (F6P), phytic acid (PhyA), α-naphthyl phosphate (α-NP), β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH), riboflavin 5′-monophosphate sodium salt hydrate (FMN), adenosine 3′,5′-cyclic monophosphate sodium salt monohydrate (c-AMP), D-glucose 6-phosphate sodium salt (G6P) (all from Sigma-Aldrich), and adenosine 5′-monophosphate sodium salt (AMP) (Fisher Scientific).

The specific activity of SapM towards the 34 substrates was determined using a 100 µl reaction mixture containing reaction buffer (50 mM Tris-Base, 150 mM NaCl, pH 7.5), 80 µM of substrate and 1 µg of protein. The reaction mixtures were incubated at 37 °C for 30 min prior to the addition of 15 µl of malachite green reagent. Subsequently, the reaction was further incubated for 15 min at room temperature (21 °C). The absorbance was read at 620 nm using a Multiskan® Spectrum spectrophotometer (Thermo Scientific). The concentration of free phosphate produced was determined using a phosphate standard curve (25–3000 pmol of Sigma-Aldrich phosphate standard solution).

Specificity activity (wild-type and mutants) towards pNPP, ATP, PI3P and PI(4,5)P₂ was evaluated with 80 µM of substrate and 0.1 µg of protein. For kinetic measurements (wild-type and mutants), we used 3–750 µM of substrate and 0.1 µg of protein. Incubation was at 37 °C for 1 h. Following steps were the same as explained above. All the experiments were performed in triplicates in 96-well microplates F-bottom clear plates in at least two separate studies.