Evolutionary analysis of the AdoMetDC family. (A) Phlyogenetic tree analysis of representative eukaryotic AdoMetDCs. (B) Partial sequence alignment of AdoMetDCs. The processing site (highlighted in gray) demarcates the serine residue (S68 in human AdoMetDC numbering; S86 in T. brucei AdoMetDC) converted to pyruvate (Py) during autocatalytic cleavage to generate the functional βα subunit. The catalytic base (C82) is also highlighted. In T. brucei AdoMetDC, the α-chain contains residues 86–370, and the smaller β-chain residues 1–85. The full sequence alignment and accession numbers are presented in SI Fig. 4.

T. brucei AdoMetDC forms a heterodimer with prozyme leading to activation of the enzyme. (A) SDS/PAGE and Western blot analysis of AdoMetDC, prozyme, and AdoMetDC/prozyme complex. Coomassie-stained gel of purified AdoMetDC (lane 1), prozyme (lane 2), and the copurified complex (lane 3). Western blot of copurified complex showing His-tagged AdoMetDC probed with α-T. brucei AdoMetDC (lane 4) and Flag-tagged prozyme probed with α-Flag antibody (lane 5). For AdoMetDC, both the α subunit of 34 kDa and 11.9 kDa β-subunit are observed on the Coomassie-stained gel, but only the α-subunit is recognized by the antibody. (B). Activation of catalysis by titration of purified recombinant prozyme (0–2 μM) into purified AdoMetDC (0.2 μM) at saturating (1 mM) AdoMet. (C) Sedimentation equilibrium by analytical ultracentrifugation of the copurified AdoMetDC/prozyme complex. Data were collected at 15,000 (○) and 20,000 (Δ) rpm at 6 μM complex. The data were globally fitted to a single ideal-species model, and the molecular mass of the species was calculated to be 80,101 Da (95% confidence interval 79,212–80,997), which corresponds well to the predicted heterodimer weight of 81,400. There was no evidence for dissociation under the experimental conditions, thus the K_d for dimer dissociation is below the level of detection of the method (K_d < 0.5 μM).

Analysis of AdoMetDC activity and expression in T. brucei lysates. (A) Northern blot analysis of mRNA (1 μg per lane) isolated from T. brucei cells for both AdoMetDC (Top) and prozyme (Middle) in procyclic form (PF), and blood form (BF) trypanosomes; tubulin (Bottom) is shown as a loading control. Molecular weight markers are given in kilobases. (B) Active-site titration of AdoMetDC in trypanosome BF cell lysates. Lysate (0.04 mg total protein; 0.01 ml at 4 mg/ml) was incubated with MDL 73811 (0–25 nM) for 1 h at 37°C. Remaining enzyme activity was determined (v_i) and compared with the uninhibited activity (v_o) in the lysate by quantitation of the amount of ¹⁴CO₂ liberated from 1-¹⁴CO₂-AdoMet. The concentration of AdoMetDC in lysates was estimated from the linear portion of the graph by the x-intercept (AdoMetDC = 6 nM). (Inset) Western blot of T. brucei AdoMetDC in PF and BF trypanosome cell lysates (0.04 mg of total protein per lane). Intensity of the 34-kDa band was compared with that measured for a titration of recombinant AdoMetDC (0.25–3 ng of protein) providing an estimate of the AdoMetDC protein concentration in the BF lysates (AdoMetDC = 5 nM). Based on the MDL 73811 titration and the Western blot analysis, the specific activity of AdoMetDC in the BF lysate can be estimated to be 0.8 s⁻¹ (1.1 μmol/min/mg) at 0.04 mM AdoMet, which would correspond to ≈3 s⁻¹ at saturating substrate (assuming the K_m reported in Table 1).