ABSTRACT
Bacterial phospholipid N-methyltransferases (Pmts) catalyze the formation of phosphatidylcholine (PC) via successive N-methylation of phosphatidylethanolamine (PE). They are classified into Sinorhizobium-type and Rhodobacter-type enzymes. The Sinorhizobium-type PmtA protein from the plant pathogen Agrobacterium tumefaciens is recruited to anionic lipids in the cytoplasmic membrane via two amphipathic helices called αA and αF. Besides its enzymatic activity, PmtA is able to remodel membranes mediated by the αA domain. According to the Heliquest program, αA- and αF-like amphipathic helices are also present in other Sinorhizobium- and Rhodobacter-type Pmt enzymes suggesting a conserved architecture of α-helical membrane-binding regions in these methyltransferases. As representatives of the two Pmt families, we investigated the membrane binding and remodeling capacity of Bradyrhizobium japonicum PmtA (Sinorhizobium-type) and PmtX1 (Rhodobacter-type), which act cooperatively to produce PC in consecutive methylation steps. We found that the αA regions in both enzymes bind anionic lipids similar to αA of A. tumefaciens PmtA. Membrane binding of PmtX1 αA is enhanced by its substrate monomethyl-PE indicating a substrate-controlled membrane association. The αA regions of all investigated enzymes remodel spherical liposomes into tubular filaments suggesting a conserved membrane-remodeling capacity of bacterial Pmts. Based on these results we propose that the molecular details of membrane-binding and remodeling are conserved among bacterial Pmts.
Subject(s)
Agrobacterium tumefaciens/enzymology , Bacterial Proteins/chemistry , Liposomes/chemistry , Methyltransferases/chemistry , Rhodobacter/enzymology , Sinorhizobium/enzymology , Agrobacterium tumefaciens/genetics , Amino Acid Sequence , Bacterial Proteins/classification , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Cell Membrane/chemistry , Cell Membrane/metabolism , Cloning, Molecular , Conserved Sequence , Escherichia coli/enzymology , Escherichia coli/genetics , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Isoenzymes/chemistry , Isoenzymes/classification , Isoenzymes/genetics , Isoenzymes/metabolism , Liposomes/metabolism , Methylation , Methyltransferases/classification , Methyltransferases/genetics , Methyltransferases/metabolism , Phosphatidylcholines/chemistry , Phosphatidylcholines/metabolism , Phosphatidylethanolamines/chemistry , Phosphatidylethanolamines/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/classification , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Rhodobacter/genetics , Sinorhizobium/genetics , Substrate SpecificityABSTRACT
The membrane lipid phosphatidylcholine (PC) is crucial for stress adaptation and virulence of the plant pathogen Agrobacterium tumefaciens. The phospholipid N-methyltransferase PmtA catalyzes three successive methylations of phosphatidylethanolamine to yield PC. Here, we asked how PmtA is recruited to its site of action, the inner leaflet of the membrane. We found that the enzyme attaches to the membrane via electrostatic interactions with anionic lipids, which do not serve as substrate for PmtA. Increasing PC concentrations trigger membrane dissociation suggesting that membrane binding of PmtA is negatively regulated by its end product PC. Two predicted alpha-helical regions (αA and αF) contribute to membrane binding of PmtA. The N-terminal helix αA binds anionic lipids in vitro with higher affinity than the central helix αF. The latter undergoes a structural transition from disordered to α-helical conformation in the presence of anionic lipids. The basic amino acids R8 and K12 and the hydrophobic amino acid F19 are critical for membrane binding by αA as well as for activity of full-length PmtA. We conclude that a combination of electrostatic and hydrophobic forces is responsible for membrane association of the phospholipid-modifying enzyme.
