A fluorescence-based assay for measuring polyamine biosynthesis aminopropyl transferase-mediated catalysis

Abstract

Polyamines are polycationic molecules that are crucial in a wide array of cellular functions. Their biosynthesis is mediated by aminopropyl transferases (APTs), which are promising targets for antimicrobial, antineoplastic, and antineurodegenerative therapies. A major limitation in studying APT enzymes, however, is the lack of high-throughput assays to measure their activity. We have developed the first fluorescence-based assay, diacetyl benzene (DAB)-APT, for the measurement of APT activity using 1,2-DAB, which forms fluorescent conjugates with putrescine, spermidine, and spermine, with fluorescence intensity increasing with the carbon chain length. The assay has been validated using APT enzymes from Saccharomyces cerevisiae and Plasmodium falciparum, and the data further validated by mass spectrometry and TLC. Using mass spectrometry analysis, the structures of the fluorescent putrescine, spermidine, and spermine 1,2-DAB adducts were determined to be substituted 1,3-dimethyl isoindoles. The DAB-APT assay is optimized for high-throughput screening, facilitating the evaluation of large chemical libraries. Given the critical roles of APTs in infectious diseases, oncology, and neurobiology, the DAB-APT assay offers a powerful tool with broad applicability, poised to drive advancements in research and drug discovery.

Keywords: 1,2-diacetyl benzene; Babesia; Plasmodium; aminopropyl transferases; drug discovery; enzyme activity; fluorescence assay; inhibition; isoindole; kinetics; polyamines; putrescine; spermidine; spermine; yeast.

Figure 1

Application of 1,2 DAB/β-ME based fluorescent assay to monitor aminopropyl transferase activity.A, schematic representation of the APT reaction and the detection of the products, spermidine or spermine, following the conversion of either putrescine or spermidine in the presence of dc-SAM, respectively, and subsequent interaction with 1,2 DAB to produce fluorescent adducts. Structures of the polyamines putrescine (PUT), spermidine (SPD), and spermine (SPM) are shown. B and C, total fluorescence intensities (λ_ex = 364 nm and λ_em = 425 nm) following incubation of 100 μM of either MTA, dc-SAM, putrescine, spermidine, or spermine with the APT reaction buffer containing 1,2-DAB in the absence (B), or presence of β-mercaptoethanol (C). The data are presented from three independent experiments performed in duplicates, and values are mean ± SD. β-ME, beta-mercaptoethanol; 1,2-DAB, 1,2-diacetyl benzene; dc-SAM, decarboxylated SAM; MTA, 5′-methylthioadenosine.

Figure 2

Changes in the ratios of putrescine-spermidine and spermidine-spermine produce quantitative changes in 1,2-DAB/β-ME fluorescence.A, putrescine, dcSAM, spermidine, and MTA were mixed in different ratios as indicated in the figure (decreasing concentration of putrescine + dcSAM and increasing concentrations of spermidine + MTA), and the total PUT + dcSAM + SPD + MTA concentration was maintained at 200 μM. The total fluorescence intensity was measured at 364 nm (excitation)/425 nm (emission) after 1 h of incubation with 1,2-DAB/β-ME. B, the net fluorescent intensity (FI) was calculated from data in Fig. 2A. A linear increase in net fluorescence intensity of spermidine with increasing concentration was observed. C, TLC showing the separation of different fractions containing decreasing concentrations of putrescine + dcSAM and increasing concentrations of spermidine + MTA as shown in the figure. D, spermidine and spermine were mixed in different ratios as indicated in the figure (decreasing concentration of spermidine + dcSAM and increasing concentrations of spermine + MTA), and the total SPD + dcSAM + SPM + MTA concentration was maintained at 200 μM. The total fluorescence intensity was measured at 364 nm (excitation)/425 nm (emission) after 1 h of incubation with 1,2-DAB/β-ME. E, the net fluorescent intensity (FI) was calculated from data in Fig. 2D. A linear increase in net fluorescence intensity of spermine with increasing concentration was observed. F, TLC showing the separation of different fractions containing decreasing concentrations of spermidine + dcSAM and increasing concentrations of spermine + MTA as shown in the figure. The data are from three independent experiments conducted in triplicates, with error bars denoting mean ± standard error. 1,2-DAB, 1,2-diacetyl benzene; dc-SAM, decarboxylated SAM; β-ME, beta-mercaptoethanol; MTA, 5′-methylthioadenosine.

Figure 3

Putrescine, spermidine, and spermine form fluorescent adducts with 1,2-DAB. Proposed mechanism of formation of fluorescent 1,3-dimethyl isoindole adducts. Formation of 1,2-DAB-polyamine adducts from 1,2-DAB and putrescine, spermidine, and spermine. 1,2-DAB, 1,2-diacetyl benzene.

Figure 4

Application of the DAB-APT fluorescence assay to determine the activity Saccharomyces cerevisiae spermidine synthase Spe3.A, schematic representation of the enzymatic reaction catalyzed by Spe3 along with and the anticipated fluorescence signals using the DAB-APT assay. B, standard curve was generated by mixing different ratios of putrescine, dcSAM, spermidine, and MTA (decreasing concentration of putrescine + dcSAM and increasing concentrations of spermidine + MTA) as indicated in the figure, and the total PUT + dcSAM + SPD + MTA concentration was maintained at 1 mM. The total fluorescence intensity was measured at 364 nm (excitation)/425 nm (emission) after 1 h of incubation with 1,2-DAB/β-ME. A linear increase in fluorescence is observed with saturation around 0.1 mM of PUT, dcSAM, and 0.4 mM of SPD + MTA. C, spermidine synthase assays were conducted using affinity-purified recombinant MBP-Spe3 (20 ng/μl), heat-inactivated MBP-Spe3 and 0.5 mM of putrescine, and dc-SAM as substrate and co-substrate, respectively, at 37 °C for 0 to 60 min. The total fluorescence intensities of the reactions catalyzed by heat-inactivated or active MBP-Spe3 at 0 min and 60 min were measured at 364 nm (excitation)/425 nm (emission) after 1 h of incubation with 1,2-DAB/β-ME buffer. D, conversion rates of the substrates putrescine and dcSAM by heat-inactivated and active MBP-Spe3 as determined by DAB assay is shown as percentage of spermidine formed, from data in Fig. 4C. Data presented as mean ± SD from three independent experiments, each conducted in triplicate. E, TLC showing the reactions performed in Fig. 4C, confirming the formation of the product spermidine by active MBP-Spe3. F, concentration of polyamines (substrate putrescine and the product spermidine) in reactions catalyzed by active and heat-denatured MBP-Spe3 after 60 min of reactions as determined using LC-MS. 1,2-DAB, 1,2-diacetyl benzene; APT, aminopropyl transferase; β-ME, beta-mercaptoethanol; MTA, 5′-methylthioadenosine; dc-SAM, decarboxylated SAM; MBP, maltose-binding protein.

Figure 5

Application of the DAB-APT fluorescence assay to determine the activity of Saccharomyces cerevisiae spermine synthase Spe4.A, schematic representation of the enzymatic reaction catalyzed by Spe4, along with the anticipated fluorescence signals using the DAB-APT assay. B, standard curve was generated by mixing different ratios of spermidine, dcSAM, spermine, and MTA (decreasing concentration of spermidine + dcSAM and increasing concentrations of spermine + MTA) as indicated in the figure, and the total SPD + dcSAM + SPM + MTA concentration was maintained at 1 mM. The total fluorescence intensity was measured at 364 nm (excitation)/425 nm (emission) after 1 h of incubation with 1,2-DAB/β-ME. A linear increase in fluorescence is observed. C, spermine synthase assays were conducted using recombinant MBP-Spe4 (20 ng/μl), heat-inactivated MBP-Spe4, and 0.5 mM of spermidine and dc-SAM as substrate and cosubstrate, respectively, at 37 °C for 0 to 60 min. The total fluorescence intensities of the reactions catalyzed by heat-inactivated or active MBP-Spe4 at 0 min and 60 min were measured at 364 nm (excitation)/425 nm (emission) after 1 h of incubation with 1,2-DAB/β-ME buffer. D, conversion rates of the substrates spermidine, and dcSAM by heat inactivated and active MBP-Spe4 is shown as percentage of spermine formed, from data Fig. 5C. Data presented as mean ± SD from three independent experiments, each conducted in triplicate. E, TLC showing the reactions performed in Fig. 5C, confirming the formation of the product spermine by active Spe4. F, concentration of polyamines (substrate spermidine and the product spermine) in reactions catalyzed by active and heat denatured Spe4 after 60 min of reaction determined using LC-MS. 1,2-DAB, 1,2-diacetyl benzene; APT, aminopropyl transferase; β-ME, beta-mercaptoethanol; MTA, 5′-methylthioadenosine; dc-SAM, decarboxylated SAM; MBP, maltose-binding protein.

Figure 6

Application of the DAB-APT fluorescence assay to determine the activity of the Plasmodium falciparum PfSPDS enzyme.A, spermidine synthase assays were conducted using affinity-purified recombinant MBP-PfSPDS (20 ng/μl), heat-inactivated MBP-PfSPDS, MBP-triple mutant PfSPDS (MBP-PfSPDS^{D127A,E147A,D196A}), and 0.5 mM of putrescine and dc-SAM as substrate and cosubstrate, respectively, at 37 °C for 0 to 60 min. The total fluorescence intensities of the above-mentioned reactions at 0 min and 60 min were measured at 364 nm (excitation)/425 nm (emission) after 1 h of incubation with 1,2-DAB/β-ME buffer. B, conversion rates of the substrates putrescine, and dcSAM by heat-inactivated, active and triple mutant MBP-PfSPDS (is shown as percentage of spermidine formed, from data in Fig. 6A. Data presented as mean ± SD from three independent experiments, each conducted in triplicate. C, spermine synthase assays were conducted using heat-inactivated or active MBP-PfSPDS, with 0.5 mM of spermidine and 0.75 mM of dc-SAM as substrate and cosubstrate, respectively, at 37 °C for 0 to 60 min. The total fluorescence intensities of the reactions catalyzed by heat-inactivated or active MBP-PfSPDS at 0 min and 60 min were measured at 364 nm (excitation)/425 nm (emission) after 1 h of incubation with 1,2-DAB/β-ME buffer. Data presented as mean ± SD from three independent experiments, each conducted in triplicate. D, TLC showing the reactions performed in Fig. 6A, confirming the formation of the product spermidine by active MBP-PfSPDS. E, TLC showing the reactions performed in Fig. 6C, showing the formation of the product spermine by active MBP-PfSPDS at 60 min. F, concentration of polyamines (substrate putrescine and the products spermidine, and spermine) in a reaction catalyzed by active, heat-denatured, and the triple mutant PfSPDS after 90 min, as determined by LC-MS. 1,2-DAB, 1,2-diacetyl benzene; APT, aminopropyl transferase; β-ME, beta-mercaptoethanol; MTA, 5′-methylthioadenosine; dc-SAM, decarboxylated SAM; MBP, maltose-binding protein; PfSPDS, P. falciparum spermidine synthase.

Figure 7

Application of the DAB-APT fluorescence assay to determine the activities and kinetics of Saccharomyces cerevisiae spermidine synthase Spe3, spermine synthase Spe4, and Plasmodium falciparum spermidine synthase.A, spermidine synthase assays were conducted using affinity-purified recombinant MBP-Spe3 (20 ng/μl), heat-denatured Spe3 (Spe3_DN), and 100 μM of putrescine and dc-SAM as substrate and cosubstrate, respectively, at 37 °C for 0 to 60 min. A parallel reaction at 4 ˚C (Spe3 4 °C) served as a control, the same reaction was also performed at 4 °C. Purified MBP-Spe4, which lacks the ability to convert putrescine to spermidine, was included as an additional control. The spermidine synthase activity of MBP-Spe3, MBP-Spe4, and respective controls is depicted as net fluorescence intensity over time. B, spermidine synthase APT activity of yeast SPE3 calculated from Fig. 7A and shown as a function of time. C and D, kinetics of the Spe3 spermidine synthase activity as a function of putrescine (C) and dc-SAM (D) concentrations. Spe3-APT assays were performed with 20 ng/μl of MBP-Spe3 and varying concentrations of either putrescine or dc-SAM at 37 °C for 60 min. E, spermine synthase assays mediated by Spe4 were performed using affinity-purified recombinant MBP-Spe4 (20 ng/μl), heat-denatured SPE4 (Spe4_DN), along with 100 μM of spermidine and dc-SAM as substrates at 37 °C for 0 to 60 min. A control reaction was conducted at 4 °C (Spe 4 °C). Spe3, which lack the ability to convert spermidine to spermine, was used as an additional control. The spermine synthase activity of MBP-Spe4, MBP-Spe3, and controls is depicted as net fluorescence intensity over time. F, spermine synthase activity of yeast Spe4 calculated from data in Fig. 7E and represented as molar amounts of spermidine formed over time. G and H, kinetics of the Spe4 spermine synthase activity as a function of spermidine (G) and dc-SAM (H) concentrations. Spe4-APT assays were performed with 20 ng/μl of MBP-Spe3 and varying concentrations of either spermidine or dc-SAM at 37 °C for 60 min. I, spermidine synthase reactions were conducted using affinity-purified recombinant MBP-PfSPDS (20 ng/μl), mutant enzymes (MBP-PfSPDS^D127A, MBP-PfSPDS^E147A, MBP-PfSPDS^{D127A,E147A,D196A}), and 100 μM of putrescine and dc-SAM as substrate and cosubstrate, respectively, at 37 °C for 0 to 60 min. The spermidine synthase activity of MBP-PfSPDS, PfSPDS mutants, and controls is depicted as net fluorescence intensity over time. J, spermidine synthase activity of PfSPDS and mutants was calculated from data in Fig. 7I and shown as a function of time. K and L, kinetics of PfSPDS spermidine synthase activity as a function of putrescine (K) or dc-SAM (L) concentrations. PfSPDS-APT assays were conducted with PfSPDS and varying concentrations of either putrescine or dc-SAM at 37 °C for 60 min. V_max and K_m values were determined using Michaelis–Menten kinetics in GraphPad Prism. Data are presented as mean ± SD from three independent experiments, each conducted in triplicate. 1,2-DAB, 1,2-diacetyl benzene; APT, aminopropyl transferase; β-ME, beta-mercaptoethanol; MTA, 5′-methylthioadenosine; dc-SAM, decarboxylated SAM; MBP, maltose-binding protein

Figure 8

Inhibition of Saccharomyces cerevisiae Spe3 and Plasmodium falciparum PfSPDS activity by 4MCHA.A, A dose-dependent decrease in the activity of MBP-Spe3 with increasing concentrations of 4MCHA (0.0195μM–100 μM). B, a dose-dependent decrease in the activity of MBP-PfSPDS with increasing concentrations of 4MCHA (0.0195μM–100 μM). The data are from three independent experiments conducted in triplicates, with error bars denoting mean ± standard error. C, TLC of the reactions performed in Fig. 8A, showing a dose-dependent decrease in the activity of Spe3. D, TLC of the reactions performed in Fig. 8B, showing a dose-dependent decrease in the activity of PfSPDS. E and F, determination of inhibition constants (Ki) values for inhibition of S. cerevisiae Spe3 (B) and P. falciparum PfSPDS (C) spermidine synthase activity by 4MCHA. All data are presented as mean ± SD from three independent experiments, each conducted in triplicate. MBP, maltose-binding protein; 4MCHA, 4-methylcyclohexylamine; PfSPDS, P. falciparum spermidine synthase.