Bioengineering SAM synthetase for the production of SAM nucleobase analogs

Abstract

S-adenosyl methionine (SAM) is a cofactor utilized in a variety of essential biochemical reactions and pathways, such as DNA, RNA and protein methylation, transsulfuration, and the biosynthesis of polyamines and vitamins such as Vitamin B1 and Vitamin B12. SAM is synthesized enzymatically from adenosine triphosphate (ATP) and L-methionine (Met) by SAM synthetase (methionine adenosyltransferase; MAT). Orthophosphate (Pi) and pyrophosphate (PPi) are both byproducts of this reaction. MAT from Escherichia coli (EcMAT) is known to be specific for ATP, whereas MAT from the thermophilic archaeon Methanocaldococcus jannaschii (MjMAT) displays activity with a variety of nucleotides (NTPs) in addition to ATP. The mechanistic role played by the nucleobase adenine in the catalytic reaction is unclear. In this project, we employ a rational engineering approach to enable EcMAT to accept NTPs other than ATP, its cognate substrate. We first corroborate results from previous studies by performing reactions for EcMAT and MjMAT with alternative nucleotides. We also examine substrate and product accommodation in the active site of EcMAT in order to determine suitable residues which upon modification could give rise to promiscuity in EcMAT. Subsequently, we inspect the effect of two distinct mutations on the substrate specificity of EcMAT. We find that the mutant EcMAT G117A demonstrates activity with ATP, GTP, and CTP, showing formation of their corresponding SAM nucleobase analogs.