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Abstract

Methylene-tetrahydropterin reductases are folded in (βα)8 barrel and catalyze the reduction of a methylene to a methyl group bound to a reduced pterin as C1 carrier in various one-carbon (C1) metabolisms. F420-dependent methylene-tetrahydromethanopterin (methylene-H4MPT) reductase (Mer) and the flavin-independent methylene-tetrahydrofolate (methylene-H4F) reductase (Mfr) use a ternary complex mechanism for the direct transfer of a hydride from F420H2 and NAD(P)H to the respective methylene group, whereas FAD-dependent methylene-H4F reductase (MTHFR) uses FAD as prosthetic group and a ping-pong mechanism to catalyze the reduction of methylene-H4F. A ternary complex structure of MTHFR is available and based on this structure, a catalytic mechanism was proposed, while no ternary complex structures of Mfr or Mer are reported. Here, Mer from Methanocaldococcus jannaschii (jMer) was heterologously produced and the crystal structures of the enzyme with and without F420 were determined. A ternary complex of jMer was modeled using a functional alignment approach based on the ternary complex structure of MTHFR and the modeled ternary complex of Mfr. Mutational analysis at the structurally conserved positions of the three reductases indicated that although these reductases share a limited sequence identity, the key catalytic glutamate residue is conserved and a common catalytic mechanism involving the formation of a 5-iminium cation of the methylene-tetrahydropterin intermediate is shared. A phylogenetic analysis indicated that the three reductases do not share one common ancestor and the conserved active site structures of the three reductases may be the result of convergent evolution.STATEMENT This work provides evidence for a common catalytic mechanism of the functional class of methylene-tetrahydropterin reductases. Despite their very low sequence identity, they share a (βα)8-barrel structure with a similar active site geometry. Phylogenetic and mutational analyses suggested that these enzymes have developed from distinct ancestors as a result of convergent evolution. This work describes an example of a catalytic mechanism that emerged independently for several times during evolution in the three domains of life.Competing Interest StatementThe authors have declared no competing interest.