Lysine 2,3-Aminomutase and a Newly Discovered Glutamate 2,3-Aminomutase Produce β-Amino Acids Involved in Salt Tolerance in Methanogenic Archaea

Abstract

Many methanogenic archaea synthesize β-amino acids as osmolytes that allow survival in high salinity environments. Here, we investigated the radical S-adenosylmethionine (SAM) aminomutases involved in the biosynthesis of Nε-acetyl-β-lysine and β-glutamate in Methanococcus maripaludis C7. Lysine 2,3-aminomutase (KAM), encoded by MmarC7_0106, was overexpressed and purified from Escherichia coli, followed by biochemical characterization. In the presence of l-lysine, SAM, and dithionite, this archaeal KAM had a k cat = 14.3 s–1 and a K m = 19.2 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. We further describe the function of MmarC7_1783, a putative radical SAM aminomutase with a ∼160 amino acid extension at its N-terminus. Bioinformatic analysis of the 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. β-Glutamate has been known to serve as an osmolyte in select methanogens for a long time, but its biosynthetic origin remained unknown until now. Thus, this study defines the biosynthetic routes for β-lysine and β-glutamate in M. maripaludis and expands the importance and diversity of radical SAM enzymes in all domains of life.

Advances in the synthesis of β-alanine

“…After the Frontiers in Bioengineering and Biotechnology frontiersin.org substrate is ingested by the engineered bacteria, it undergoes a series of metabolic processes and eventually enters the TCA cycle in the form of PEP or acetyl-CoA. Currently, it is common to overexpress or (and) optimize the expression of the ppc gene using a synthetic promoter and RBS sequences to efficiently convert PEP to oxaloacetate, which significantly increases β-alanine production (Zou et al, 2020;Tunckanat et al, 2022;Dobrowolski et al, 2020;Parthasarathy et al, 2019;Piao et al, 2019;Song et al, 2015).…”

Biochemical and genetic studies define the functions of methylthiotransferases in methanogenic and methanotrophic archaea

“…A Bacillus subtilis strain lacking the MtaB-encoding gene (B. subtilis 168 ΔyqeV::kan, BKK25430) (Koo et al, 2017) was obtained from the Bacillus Genetic Stock Center and grown in LB medium with 7.5 μg/mL kanamycin. Methanococcus maripaludis S2 Mm902 (Walters et al, 2011;Tunckanat et al, 2022) was obtained from the laboratory of Dr. John Leigh (University of Washington) and grown in McCas medium (Moore and Leigh, 2005) with H 2 /CO 2 (80/20, 40 psi). M. maripaludis S2 Mm902 was constructed as reported in Walters et al (2011), except the ORF1 Methylthiotransferase reaction schemes.…”