Biochemical Characterization of Aminomutases Involved in Salt Tolerance in Methanogenic Archaea

Abstract

Lysine-2,3-aminomutase (LAM) catalyzes the conversion of L-α-lysine to L-β-lysine, which entails the transfer of an α-amino group to the b-carbon alongside the migration of a β-hydrogen to the α-carbon. This enzyme plays a role in combating salt stress in methanogenic archaea by carrying out the first step in the biosynthesis of Ne-acetyl-β-lysine, a well-studied osmolyte synthesized and accumulated in methanogens. LAM is of special interest as it belongs to the radical S-adenosyl-L-methionine (SAM) superfamily of enzymes which perform diverse and complex chemistry that often produces valuable compounds. Radical SAM enzymes utilize a reduced [4Fe-4S] cluster to initiate the homolytic cleavage of SAM, yielding a highly reactive 5’-deoxyadenosyl radical that then abstracts a hydrogen atom from an otherwise unreactive substrate. Although the bacterial LAM involved in lysine degradation in Clostridium subterminale has been well-characterized, the archaeal LAM involved in osmolyte biosynthesis has never been studied in vitro. Here, we report on the recombinant expression, purification, and enzymatic properties of LAM from Methanococcus maripaludis. The gene encoding LAM from M. maripaludis C7 (MmarC7_0106) was cloned and the his-tagged protein was overexpressed in Escherichia coli. After purification by metal-affinity chromatography under strictly anaerobic conditions, the protein was brown in color, had UV-Vis spectrum characteristics of a [4Fe-4S] cluster that was capable of being reduced to an active state, and had 3.9 mol of iron per monomer. A wide ranged temperature profile study revealed that enzymatic activity was highest at 37°C. The enzyme reaction in the presence of L-lysine, SAM, and dithionite at 37°C had a Kcat = 0.42 s-1 and a Km = 17.8 mM. Finally, alternative substrates for the aminomutation reaction were investigated. There exists another gene annotated as LAM in M. maripaludis C7 due to its close sequence similarity with the bacterial LAM; however, this gene (MmarC7_1783) lacks lysine binding residues and has a 164 amino acid N-terminal extension and as such is referred to as a “long-LAM”. The presence of a “long-LAM” such as MmarC7_1783 in a methanogen is not uncommon as there are several annotated in thermophilic methanogenic species that are known to solely use β-glutamate, another beta-amino acid, as their osmolyte. Given that the only known pathway for the biosynthesis of β-glutamate is through the use of a glutamate-2,3-aminomutase (GAM) and that these thermophilic methanogens do not have an annotated GAM, we proposed that these long-LAMs were in fact GAM which we have now confirmed through heterologous expression studies. Thus, in addition to the biochemical characterization of an archaeal LAM (MmarC7_0106) described above, we have determined that long-LAMs are in fact GAMs required in the biosynthesis of an alternative osmolyte used for survival in high salt concentrations by halotolerant methanogenic archaea.