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Isolation and functional expression in Escherichia coli of a gene encoding phosphatidylethanolamine methyltransferase
V Arondel1, C Benning, C R Somerville
1Michigan State University, Department of Energy Plant Research Laboratory, East Lansing 48824-1312.
Abstract:
Phosphatidylcholine is a major component of membranes in most eukaryotes, but it is found only in a small number of bacteria, where it is synthesized by N-methylation of phosphatidylethanolamine. In yeast and other fungi the methylation of phosphatidylethanolamine to phosphatidylcholine proceeds in two steps: the methylation of phosphatidylethanolamine by phosphatidylethanolamine methyltransferase followed by the methylation of monomethylphosphatidylethanolamine by phospholipid methyltransferase. Here we describe the isolation of two allelic phosphatidylcholine-deficient mutants of Rhodobacter sphaeroides which are unable to methylate phosphatidylethanolamine, monomethylphosphatidylethanolamine, or dimethylphosphatidylethanolamine. A DNA fragment containing a gene designated pmtA, which encodes a 22.9-kDa protein, was found to complement both mutants. Expression of this gene in Escherichia coli, which normally lacks phosphatidylcholine or methylated derivatives of phosphatidylethanolamine, resulted in the formation of phosphatidylcholine. A protein extract derived from the E. coli strain expressing the pmtA gene was able to convert phosphatidylethanolamine, mono- and dimethylphosphatidylethanolamine into phosphatidylcholine. Based on these data we conclude that the product of the pmtA gene catalyzes a sequence of three chemically distinct, methylation reactions beginning with phosphatidylethanolamine and leading to the formation of phosphatidylcholine in R. sphaeroides.
Insights
Researchers identified the pmtA gene in Rhodobacter sphaeroides, which encodes a protein responsible for synthesizing phosphatidylcholine through sequential methylation of phosphatidylethanolamine. This finding elucidates a key bacterial membrane lipid biosynthesis pathway.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Phosphatidylcholine is a vital membrane component in eukaryotes but rare in bacteria.
- Bacterial synthesis often involves N-methylation of phosphatidylethanolamine.
- Fungi utilize a two-step methylation process involving specific methyltransferases.
Purpose of the Study:
- To investigate the genetic basis of phosphatidylcholine synthesis in Rhodobacter sphaeroides.
- To identify the gene responsible for methylating phosphatidylethanolamine and its derivatives.
Main Methods:
- Isolation and characterization of phosphatidylcholine-deficient mutants in Rhodobacter sphaeroides.
- Complementation of mutants using a DNA fragment containing the pmtA gene.
- Heterologous expression of pmtA in Escherichia coli to assess functional complementation.
- Biochemical assays using protein extracts to confirm enzymatic activity.
Main Results:
- Two allelic mutants deficient in methylating phosphatidylethanolamine, monomethylphosphatidylethanolamine, and dimethylphosphatidylethanolamine were isolated.
- A gene, pmtA, encoding a 22.9-kDa protein, was identified and complemented both mutants.
- Expression of pmtA in E. coli led to the production of phosphatidylcholine.
- Protein extracts from E. coli expressing pmtA demonstrated the ability to convert phosphatidylethanolamine and its methylated derivatives into phosphatidylcholine.
Conclusions:
- The pmtA gene product catalyzes a sequential three-step methylation reaction.
- This pathway leads to the formation of phosphatidylcholine from phosphatidylethanolamine in Rhodobacter sphaeroides.
- The study identifies a novel bacterial enzyme involved in membrane lipid biosynthesis.