S -Adenosylmethionine-Binding Properties of a Bacterial Phospholipid N -Methyltransferase

Abstract

The presence of the membrane lipid phosphatidylcholine (PC) in the bacterial membrane is critically important for many host-microbe interactions. The phospholipid N-methyltransferase PmtA from the plant pathogen Agrobacterium tumefaciens catalyzes the formation of PC by a three-step methylation of phosphatidylethanolamine via monomethylphosphatidylethanolamine and dimethylphosphatidylethanolamine. The methyl group is provided by S-adenosylmethionine (SAM), which is converted to S-adenosylhomocysteine (SAH) during transmethylation. Despite the biological importance of bacterial phospholipid N-methyltransferases, little is known about amino acids critical for binding to SAM or phospholipids and catalysis. Alanine substitutions in the predicted SAM-binding residues E58, G60, G62, and E84 in A. tumefaciens PmtA dramatically reduced SAM-binding and enzyme activity. Homology modeling of PmtA satisfactorily explained the mutational results. The enzyme is predicted to exhibit a consensus topology of the SAM-binding fold consistent with cofactor interaction as seen with most structurally characterized SAM-methyltransferases. Nuclear magnetic resonance (NMR) titration experiments and 14 C-SAM-binding studies revealed binding constants for SAM and SAH in the low micromolar range. Our study provides first insights into structural features and SAM binding of a bacterial phospholipid N-methyltransferase.

Discovery of Small Molecules that Activate RNA Methylation through Cooperative Binding to the METTL3-14-WTAP Complex Active Site

“…Sinefungin expectedly inhibited the METTL3-14-WTAP activity, with an IC 50 of 2.36 mM ( Figure 4C). The observed IC 50 concentrations of SAM, SAH, and sinefungin correspond to those inhibitory IC 50 concentrations as reported in the literature for these compounds on different methyltransferases (Aktas et al, 2011).…”

Enzymatic properties and substrate specificity of a bacterial phosphatidylcholine synthase

“…To study choline binding of purified Pcs we used a filter‐binding assay with 14 C‐labeled choline, as described previously . Choline binding was observed only in the presence of both CDP–DAG and manganese (Fig.…”

“…Choline‐binding assays were carried out as described earlier . Briefly, 30 μ m of recombinant Pcs protein was incubated with 90 μ m of choline‐[methyl‐ 14 C]‐chloride (55 mCi·mmol −1 and 0.1 mCi·mL −1 ) in binding buffer (100 m m Tris/HCl, pH 8.0) for 5 min at 30 °C (total assay volume 50 μL).…”

SAMbinder: A Web Server for Predicting S-Adenosyl-L-Methionine Binding Residues of a Protein From Its Amino Acid Sequence

“…Therefore, there is a requirement of automated computational methods that can identify the residues playing an essential role in protein functions. Protein-ligand interaction has been recognized as one of the important functions which play a vital function in all biological processes (Agrawal et al, 2019e). In the past, considerable efforts have been made to develop tools that can identify the ligand-interacting residues in a protein (Sousa et al, 2006).…”