Nucleotide sequence and developmental expression of Acanthamoeba S-adenosylmethionine synthetase gene

We have isolated and characterized a cDNA (cDNA1) from an Acanthamoeba cDNA library encoding the enzyme S-adenosylmethionine (SAM) synthetase (ATP: L-methionine S-adenosyltransferase; EC 2.5.1.6). The nucleotide sequence exhibits about 61-73% overall similarity to the corresponding gene of other organisms. The cDNA displays extreme codon bias with a preference for C or G in the third position. A putative initiation site and an ATP-binding site are identified. An amino acid content of 388 and a molecular mass of about 44000 Daltons are deduced for the enzyme. Putative phosphorylation sites which might be involved in regulation of the enzyme are revealed. The cDNA was expressed in Escherichia coli BL21(DE3), and the identity of the protein product confirmed by Western blotting analysis. Northern analyses of the expression of the Acanthamoeba SAM synthetase gene during development revealed a pronounced reduction in the level of transcripts as amoebae converted to cysts.