In vitro characterization of the enzyme properties of the phospholipid N-methyltransferase PmtA from Agrobacterium tumefaciens

Abstract

Agrobacterium tumefaciens requires phosphatidylcholine (PC) in its membranes for plant infection. The phospholipid N-methyltransferase PmtA catalyzes all three transmethylation reactions of phosphatidylethanolamine (PE) to PC via the intermediates monomethylphosphatidylethanolamine (MMPE) and dimethylphosphatidylethanolamine (DMPE). The enzyme uses S-adenosylmethionine (SAM) as the methyl donor, converting it to S-adenosylhomocysteine (SAH). Little is known about the activity of bacterial Pmt enzymes, since PC biosynthesis in prokaryotes is rare. In this article, we present the purification and in vitro characterization of A. tumefaciens PmtA, which is a monomeric protein. It binds to PE, the intermediates MMPE and DMPE, the end product PC, and phosphatidylglycerol (PG) and phosphatidylinositol. Binding of the phospholipid substrates precedes binding of SAM. We used a coupled in vitro assay system to demonstrate the enzymatic activity of PmtA and to show that PmtA is inhibited by the end products PC and SAH and the antibiotic sinefungin. The presence of PG stimulates PmtA activity. Our study provides insights into the catalysis and control of a bacterial phospholipid N-methyltransferase.

FIG. 1.

Phospholipid N-methylation pathway of PC biosynthesis in bacteria. Phospholipid N-methyltransferase(s) (Pmt) catalyzes the three-step methylation of PE to PC via MMPE and DMPE. DAG, diacylglycerol.

FIG. 2.

Purification of recombinant PmtA. (A) SDS-PAGE of Ni-IDA purification of N-terminal His₆-tagged PmtA (His^PmtA). The fractions loaded were P, pellet; C, crude extract; W, wash fraction; E1 and E2, elution fractions 1 and 2; M, BenchMark protein standard (Invitrogen). The two right lanes (marked as “blot”) show Western blot analyses of E1 and E2, using anti-Penta-His HRP-coupled antibody (Qiagen). (B) Size exclusion chromatography of Ni-IDA-purified PmtA. The positions of two standard proteins (chymotrypsinogen, 25 kDa; ovalbumine, 43 kDa) and the void volume (vv) are indicated. The peak fractions 1 and 2 were analyzed by SDS-PAGE as depicted on the right. mAU, milli-absorbance units.

FIG. 3.

Initial characterization of PmtA activity. The assay was carried out in the absence (−) or presence (+) of recombinant PmtA. PmtA activity was analyzed with different phospholipid substrates (E. coli lipids [Ec], PE, MMPE, or DMPE). The products were extracted and separated by one-dimensional TLC. Phospholipid products were specifically stained with molybdenum blue. Barely detectable lipids are marked with arrows.

FIG. 4.

Protein lipid overlay assay. His₆-tagged PmtA (4 nmol) was incubated with nitrocellulose strips carrying equal amounts (14 nmol) of various phospholipids (PE, MMPE, DMPE, and PC) (A) or serial dilutions of PG and PI (0 to 14 nmol) (B). Bound protein was detected with anti-Penta-His HRP-coupled antibody (Qiagen). “Blank” is a chloroform-methanol-water (1:2:0.8) solvent. (C) PmtA activity in the presence (+) or absence of PG (12.5 μg). Assays contained 50 μg of PE, MMPE, or DMPE and 166 μM SAM. The enzymatic products were analyzed by one-dimensional TLC. Lipid products were visualized with molybdenum blue staining. Barely detectable lipids are marked with arrows.

FIG. 5.

Radioactive SAM binding assay. (A) SAM binding activity was analyzed with 0.4 nmol of recombinant PmtA and 2.5 μCi (0.6 μM SAM) of S-adenosyl-l-[methyl-³H]methionine (³H-SAM; 81.9 Ci/mmol) in the presence (+) or absence (−) of 300 μM of the phospholipid (PL) PE, MMPE, DMPE, PC, or PG. The displacement assay contained, in addition, 500-fold unlabeled (“cold”) SAM (third column). (B) Competitive displacement of radiolabeled SAM by unlabeled SAM (0 to 200 μM). The assay mixture contained 0.4 nmol of recombinant PmtA, 300 μM PE, and 2.5 μCi (0.6 μM SAM) of S-adenosyl-l-[methyl-³H]methionine (81.9 Ci/mmol). One hundred percent S-adenosyl-l-[methyl-³H]methionine corresponds to 477 × 10³ dpm.

FIG. 6.

Displacement of radiolabeled SAM by SAH and sinefungin. (A) Chemical structures of SAM, SAH, and sinefungin. (B) Recombinant PmtA (0.4 nmol) was assayed for SAM binding activity in the presence of S-adenosyl-l-[methyl-³H]methionine (³H-SAM; 2,5 μCi) and PE (300 μM) and various concentrations (0 to 200 μM) of unlabeled SAH or sinefungin. One hundred percent S-adenosyl-l-[methyl-³H]methionine corresponds to 221 × 10³ dpm in the SAH experiment and 252 × 10³ dpm in the sinefungin experiment. (C) Inhibition of in vitro PmtA activity by SAH and sinefungin. PmtA activity was analyzed as previously described with E. coli lipids and 166 μM SAM (control). An inhibition assay was performed in the presence of 166 μM (1) and 332 μM (2) of SAH and sinefungin, respectively. Enzymatic products were analyzed by one-dimensional TLC. Lipid products were visualized with molybdenum blue staining.

FIG. 7.

PmtA activity in enzyme-coupled SAM 265 assay. (A) Principle of the assay used to detect SAM-dependent methyltransferase activity according to Dorgan et al. (11). A detailed description is given in the Materials and Methods section. Nu, nucleophile. (B) Analysis of PmtA activity with natural substrate lipids isolated from A. tumefaciens ΔpmtA or ΔpmtA Δpcs mutant. Membrane lipids were isolated and separated by one-dimensional TLC. Phospholipids were specifically stained with molybdenum blue (inset). Reaction mixtures (200 μl) contained 0.01 μM adenine deaminase, 0.1 μM SAH nucleosidase, 110 μM SAM, 0.02% (wt/vol) Triton X-100, and lipids from a 2-ml culture of the A. tumefaciens ΔpmtA or ΔpmtA Δpcs mutant as the substrate. Reactions were started by the addition of 2 μM PmtA. Absorbance changes were measured at 265 nm with a 96-well plate reader (μQuant; BioTek). (C) Analysis of PmtA activity with PE micelles as substrate. Assays were performed as described for panel B with 0 to 800 μM (0 to 71 mol%) commercially available PE as the substrate. (D and E) Influence of PC and PG on PmtA activity. Assays were performed as described for panel B with 150 μM micellar PE (32 mol%) (D) or 150 μM liposomal PE (E) either in the absence (PE alone) or presence (PE+PG, PE+PC) of 60 μM or 150 μM PC or 60 μM PG. PmtA (3 μM) was used to start the assay.