Engineering a flexible loop in S-adenosyl- L -methionine synthetase enables production of SAM nucleobase analogues with selective biochemical and cellular activity

S -adenosyl- L -methionine (SAM), an essential cofactor in all forms of life, is synthesized by the enzyme methionine adenosyltransferase (MAT) from methionine and ATP. The adenine moiety in SAM appears to have no direct function in catalysis, and some MAT homologs can utilize natural nucleotide triphosphates in vitro , producing the corresponding SAM nucleobase analogues. However, the molecular determinants of nucleotide choice of the MAT enzyme and the cellular significance of the nucleobase in SAM are unclear. In this study, using structure- and bioinformatics-guided mutagenesis, we identify a flexible active-site loop as a major determinant of nucleotide specificity in MAT. Loop mutations and loop swaps convert ATP-selective Escherichia coli MAT into variants that accept GTP, CTP, and UTP, enabling enzymatic synthesis and purification of S-guanosyl-, S-cytosyl-, and S-uracyl-L-methionine. Further, we show that these analogues partially rescue the growth of an E. coli SAM auxotroph under SAM-limited growth conditions. Biochemical assays show that the analogues bind the tested SAM-utilizing enzymes; they serve as substrates for E. coli SAM decarboxylase but do not support detectable methyl transfer by E. coli DNA adenine methyltransferase. These results establish the flexible loop as a gatekeeper of MAT nucleotide specificity and show that this loop can be engineered to produce SAM analogues which can selectively participate in downstream cellular metabolism.