Lysine‐2,3‐aminomutase and a newly discovered glutamate‐2,3‐aminomutase produce β‐amino acids to overcome salt stress in methanogenic archaea

Abstract

Many methanogenic archaea synthesize N ε ‐acetyl‐β‐lysine and/or β‐glutamate as osmolytes that allow survival in high salinity environments. Methanococcus maripaludis C7 contains two genes, MmarC7_0106 and MmarC7_1783, encoding radical S‐adenosylmethionine(SAM) enzymes with sequence similarity to lysine‐2,3‐aminomutase (KAM). MmarC7_0106 is immediately upstream of an acetyltransferase and was expected to be the KAM involved in the biosynthesis of N ε ‐acetyl‐β‐lysine. To confirm this, the gene was cloned and expressed with a hexahistidine tag in E. coli, followed by anaerobic purification and biochemical characterization. Although the bacterial KAMs involved in lysine degradation in Clostridium subterminale and a post‐translational modification of elongation factor P in E. coli have been well‐characterized, the archaeal KAM involved in compatible solute biosynthesis had never been studied in vitro before. In the presence of L‐lysine, SAM, and dithionite at 37°C, this archaeal KAM had a k cat = 0.42 s ‐1 and a K m = 17.8 mM. The product was shown to be 3(S)‐β‐lysine, which is like the well‐characterized Clostridium KAM as opposed to the E. coli KAM that produces 3(R)‐β‐lysine. The archaeal KAM was highly specific for L‐lysine, with no activity observed with D‐lysine or most other amino acids. However, low aminomutase activity was observed with L‐arginine. We further report the function of MmarC7_1783, a putative radical SAM aminomutase with a ~160 amino acid extension at its N‐terminus. Bioinformatic analysis of possible substrate binding residues suggested a function as glutamate‐2,3‐aminomutase, which was confirmed here through heterologous expression in a methanogen followed by detection of β‐glutamate in cell extracts. Finally, we investigated the differences in amino acid compatible solute usage amongst M. maripaludis C7 (contains both aminomutase genes) and M. maripaludis S2 (contains only the canonical KAM gene) in different salt concentrations. Taken together, this work defines the biosynthetic routes for β‐amino acids in methanogenic archaea and expands the importance and diversity of radical SAM enzymes in all domains of life.