Exploring the nucleotide promiscuity of Escherichia coli methionine adenosyltransferase mutants

Abstract

The cofactor S-adenosyl methionine (SAM) is a critical player in cellular metabolism, involved in processes like methylating proteins and nucleic acids and biosynthesizing polyamines and specific vitamins. Its role as a versatile cofactor makes the in vivo generation of its analogs highly desirable, especially for designing biorthogonal metabolic pathways inside the cell. SAM is synthesized in the cell from adenosine–5’-triphosphate (ATP) and L-methionine by the enzyme methionine adenosyltransferase (MAT), also known as SAM synthetase. MAT is a cytosolic enzyme with access to additional non-cognate nucleoside-5’-triphosphates (NTPs), further motivating investigations into expanding the nucleotide substrate scope of the enzyme to synthesize nucleobase analogs of SAM (SNMs). In this study, we have attempted to enhance the nucleotide promiscuity of the MAT from Escherichia coli (EcMAT). We implemented a rational design approach where EcMAT was mutated based on differences between its active site and that of an NTP-promiscuous homolog of MAT from Methanocaldococcus jannaschii. We observe that all the seven EcMAT variants we characterized formed SAM in vitro, but only one – I102L EcMAT exhibited robust in vitro gains in activity with guanosine-5’-triphosphate (GTP) and uridine-5’-triphosphate (UTP). All the mutants retained SAM synthetase activity in vivo when heterologously expressed inside E. coli. The I102L mutant, when complemented with an E. coli metK (gene encoding for MAT) knockout strain, also catalyzed the formation of detectable amounts of S-guanosyl methionine (SGM) inside the organism. These results provide insights for designing novel mutagenesis-based strategies to improve EcMAT's nucleotide promiscuity. The study also provides the first evidence for the formation of SNMs inside a microbial cell.