Biology subjects

Shima, S.

Publications and source records attributed to Shima, S..

3 recordsLinked to original sources

bioRxiv2023

Convergent evolution of (beta alpha)8-barrel fold methylene-tetrahydropterin reductases utilizing a common catalytic mechanism

Methylene-tetrahydropterin reductases are folded in ({beta})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. STATEMENTThis 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 ({beta})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.

biochemistry↗

bioRxiv2023

Sensitivity-enhanced magnetic resonance reveals hydrogen intermediates during active -hydrogenase catalysis

Molecular hydrogen (H2) is considered an eco-friendly future energy-carrier and an alternative to fossil fuel1 and thus, major efforts are directed towards identifying efficient and economical hydrogen catalysts.2,3 Efficient hydrogen catalysis is used by many microorganisms, some of them producing H2 from organic materials and others consuming it.4-6 To metabolize H2, these microorganisms use enzymes called hydrogenases.7,8 For the future development of efficient catalysts a detailed analysis of the catalytic mechanisms of such hydrogenases is required and existing analytical techniques could not provide a full understanding.9 Consequently, new analytical technologies are of utmost importance to unravel natures blueprints for highly efficient hydrogen catalysts. Here, we introduce signal-enhanced or hyperpolarized, nuclear magnetic resonance (NMR) to study hydrogenases under turnover conditions. So far undiscovered hydrogen species of the catalytic cycle of [Fe]-hydrogenases, are revealed and thus, extend the knowledge regarding this class of enzymes. These findings pave new pathways for the exploration of novel hydrogen metabolisms in vivo. We furthermore envision that the results contribute to the rational design of future catalysts to solve energy challenges of our society.

biophysics↗

bioRxiv2022

Crystal structure of FAD-independent methylene-tetrahydrofolate reductase from Mycobacterium hassiacum

FAD-independent methylene-tetrahydrofolate (methylene-H4F) reductase (Mfr), recently identified in mycobacteria, catalyzes the reduction of methylene-H4F to methyl-H4F with NADH as hydride donor by a ternary complex mechanism. This biochemical reaction corresponds to that of the ubiquitous FAD-dependent methylene-H4F reductase (MTHFR), although the latter uses a ping-pong mechanism with FAD as prosthetic group. Comparative genomics and genetic analyses indicated that Mfr is indispensable for the growth of Mycobacterium tuberculosis, which lacks the MTHFR-encoding gene. Thus, Mfr is an excellent target enzyme for the design of antimycobacterial drugs. Here, we report the heterologous production, enzymological characterization and the crystal structure of Mfr from the thermophilic mycobacterium M. hassiacum (hMfr), which shows 78% sequence identity to Mfr from M. tuberculosis. Although hMfr and MTHFR show very low sequence identity and different catalytic mechanisms, their tertiary structures are highly similar, which suggests a divergent evolution of Mfr and MTHFR from a common ancestor. Most of the important active-site residues of MTHFR are conserved and equivalently positioned in the tertiary structure of hMfr. The Glu9Gln variant of hMfr exhibits a drastic reduction of the catalytic activity, which supports the predicted function of the glutamate residue as proton donor in both Mfr and MTHFR. The predicted nicotinamide binding site of hMfr is substantially narrower than the isoalloxazine binding site of MTHFR, which may reflect an evolutional adaptation to the different sizes of the coenzymes.

biochemistry↗