Chemical & Enzymatic Synthesis of S-adenosylmethionine nucleoside analogs (SNM) using Escherichia coli methionine adenosyltransferase (EcMAT) mutants to probe nucleotide promiscuity.

Abstract

S-adenosyl methionine (SAM) is the second-most-utilized cofactor in cellular reactions. It is the major methyl donor for biomolecules, including proteins, DNA, and RNA, etc. Besides this of SAM is also involved in different reactions; for example, the amine is used in the synthesis of DAPA, which is further utilized in biotin synthesis, making it highly versatile. These features make it highly coveted, thereby laying the foundation for analog-based studies to investigate enzyme mechanisms and other cellular pathways. Methionine adenosyltransferase (MAT) is the enzyme that synthesizes SAM by utilizing adenosine-5’- triphosphate (ATP) and methionine as the substrate. The product inhibition observed during synthesis has been a major concern, motivating the investigation of engineering MAT variants via site-directed mutagenesis to reduce it. Further, it is known that MAT is a cytosolic protein that binds other non-cognate NTPs yet is specific for ATP, which motivates the investigation of nucleotide-based substrate promiscuity to synthesize these SAM nucleobase analogs, called SNMs. In this study, we sought to identify MAT variants that reduced product inhibition in Escherichia coli. Additionally, attempts were made to probe for nucleotide promiscuity using mutations that target product inhibition, as these mutations reduce interactions in the enzyme pocket by enlarging it or increasing protein flexibility, thereby potentially accommodating other NTPs. Among all the mutants purified and characterized, Q21R/I303V is the best performing mutant in terms of product formation followed by I303V as reported in the literature. Amongst all I64V/L185V/I303V, I303V, and Q21R/N103A mutations show very little promiscuity toward other NTPS, though a decrease in activity for ATP was observed for these mutants. An attempt was made to incorporate the promiscuous mutant EcMAT N103A, previously made in the lab, into the chromosomal DNA of the Escherichia coli MG1655 strain using homologous recombination. The strain carrying the mutation has not yet been characterized for SNM production in vivo. But this study will lay the groundwork for exploring product inhibition and promiscuity in parallel to identify a more efficient mutant. This recombinant strain can provide us with insights into the utilization and in vivo biochemistry of these SNMs.