The thiamine biosynthetic enzyme ThiC catalyzes multiple turnovers and is inhibited by S-adenosylmethionine (AdoMet) metabolites

Abstract

ThiC (4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase; EC 4.1.99.17) is a radical S-adenosylmethionine (AdoMet) enzyme that uses a [4Fe-4S](+) cluster to reductively cleave AdoMet to methionine and a 5'-deoxyadenosyl radical that initiates catalysis. In plants and bacteria, ThiC converts the purine intermediate 5-aminoimidazole ribotide to 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate, an intermediate of thiamine pyrophosphate (coenzyme B1) biosynthesis. In this study, assay conditions were implemented that consistently generated 5-fold molar excess of HMP, demonstrating that ThiC undergoes multiple turnovers. ThiC activity was improved by in situ removal of product 5'-deoxyadenosine. The activity was inhibited by AdoMet metabolites S-adenosylhomocysteine, adenosine, 5'-deoxyadenosine, S-methyl-5'-thioadenosine, methionine, and homocysteine. Neither adenosine nor S-methyl-5'-thioadenosine had been shown to inhibit radical AdoMet enzymes, suggesting that ThiC is distinct from other family members. The parameters for improved ThiC activity and turnover described here will facilitate kinetic and mechanistic analyses of ThiC.

Keywords: Enzyme Inhibitors; Iron-Sulfur Protein; Radicals; S-adenosylmethionine (AdoMet); Thiamine; Thiamine Biosynthesis.

FIGURE 1.

ThiC reaction.

FIGURE 2.

ThiC undergoes steady-state turnover. ThiC (0.55-nmol monomer) was incubated with flavoprotein reductase (0.5 nmol), flavodoxin A (1 nmol), NADPH (0.8 mm), AdoMet (100 μm), AIR (100 μm), and MTAN as indicated (0.1 nmol) at 37 °C. Each data point represents the mean ± S.D. of two replicates from a single experiment. The data were fit to a first-order rate equation, and the 95% confidence intervals of the regression analysis are represented by dotted lines.

FIGURE 3.

Metabolite inhibitors of ThiC activity. ThiC (0.55-nmol monomer) was preincubated with flavoprotein reductase (0.5 nmol), flavodoxin A (1 nmol), NADPH (0.8 mm), and potential inhibitor (0.5 mm) for 10 min at room temperature. Then AdoMet (100 μm) and AIR (100 μm) were added to initiate the reactions, which were incubated at 37 °C for 30 min. Data represent the mean ± S.D. of two replicates. The average is significantly different from the average with no inhibitor, as determined by an unpaired Student's t test (*, p < 0.05). 2′-DOA, 2′-deoxyadenosine; HCy, homocysteine; AIRs, 5-aminoimidazole riboside; AICARs, aminoimidazole carboxamide riboside; CAIRs, 5-amino-4-imidazolecarboxylic acid riboside.

FIGURE 4.

SAH inhibits ThiC competitively with respect to AdoMet. ThiC (0.55-nmol monomer) was preincubated with flavoprotein reductase (0.5 nmol), flavodoxin A (1 nmol), NADPH (0.8 mm), AIR (100 μm), and SAH (0, 10, 25, or 50 μm) for 10 min at room temperature. Then AdoMet (25–150 μm) was added to initiate the reactions, which were incubated at 37 °C for 20 min. The data were fit to Equation 4 by non-linear regression, constraining K_m = 17 μm.

FIGURE 5.

Cooperative inhibition by 5′-DOA and Met. ThiC (0.55-nmol monomer) was preincubated with flavoprotein reductase (0.4 nmol), flavodoxin A (1 nmol), NADPH (0.8 mm), 5′-DOA (0–500 μm), and Met (0–1000 μm) for 10 min at room temperature. Then AdoMet (100 μm) and AIR (100 μm) were added to initiate the reactions, which were incubated at 37 °C for 20 min. The data were fit to Equation 5 by non-linear regression, constraining K_m = 17 μm and [AdoMet] = 100 μm.

FIGURE 6.

Adenosine is uncompetitive with AdoMet inhibiting ThiC. A, ThiC (0.55-nmol monomer) was preincubated with flavoprotein reductase (0.5 nmol), flavodoxin A (1 nmol), NADPH (0.8 mm), AIR (100 μm), and adenosine (0, 100, 250, or 400 μm) for 10 min at room temperature. Then AdoMet (AdoMet) (25–150 μm) was added to initiate the reactions, which were incubated at 37 °C for 20 min. The data were fit to Equation 6 by non-linear regression, constraining K_m = 17 μm and V_max = 0.1128 nmol HMP/nmol ThiC/min.