UCLA

S-adenosylmethionine (SAM) is a universal methyl donor required for numerous essential biochemical reactions, including DNA methylation, glutathione and polyamine synthesis, and phospholipid metabolism. SAM plays a critical role in linking the cell’s metabolic state to its diverse functions, and its dysregulation in the liver has been implicated in metabolic diseases, including metabolic dysfunction-associated steatotic liver disease, which manifests as liver steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma. To investigate SAM’s role in cellular and subcellular metabolism, we developed a genetically encoded SAM manipulator, termed

“SAMulator,” by fusing methionine adenosyltransferase (MAT) with an Xpress tag and mScarlet fluorescent protein, enabling simultaneous expression detection and enzymatic activity in mammalian cells. A screen of six candidate SAMulators, each containing MATs from different species, identified SAMulator 0m (derived from Mus musculus) as a potent enhancer of SAM production. In contrast, SAMulator 3, derived from Plasmodium falciparum, expressed well but lacked measurable activity, indicating the need for optimization. Unlike the mouse-derived MAT, which is subject to complex regulation and potential inhibition in mammalian cells, we hypothesized that the protozoan-derived MAT used in SAMulator 3 is less regulated, allowing for more consistent and controlled SAM production. To improve SAMulator 3 function, we introduced modifications, including flexible linker redesign and redox-regulating mutations (C113S, C187S), guided by previous biochemical studies. Further refinement through subcellular targeting and disruption of a predicted nuclear export signal (L343A) enhanced its compartmental localization. By refining SAMulator 3 through site-directed mutagenesis, we aim to develop a precise tool to manipulate SAM levels in distinct subcellular compartments in mammalian cells. This tool will then be utilized to investigate SAM's role in various cellular processes, paving the way for targeted metabolic therapies.