Malonyl-CoA reductase (MCR) is a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent bi-functional enzyme that performs alcohol dehydrogenase and aldehyde dehydrogenase (CoA-acylating) activities in the N- and C-terminal fragments, respectively. It catalyzes the two-step reduction of malonyl-CoA to 3-hydroxypropionate (3-HP), a key reaction in the autotrophic CO₂ fixation cycles of Chloroflexaceae green non-sulfur bacteria and the archaea Crenarchaeota. However, the structural basis underlying substrate selection, coordination, and the subsequent catalytic reactions of full-length MCR is largely unknown. For the first time, we here determined the structure of full-length MCR from the photosynthetic green non-sulfur bacterium Roseiflexus castenholzii (RfxMCR) at 3.35 Å resolution. Furthermore, we determined the crystal structures of the N-terminal and C-terminal fragments bound with reaction intermediates NADP⁺ and malonate semialdehyde (MSA) at 2.0 Å and 2.3 Å, respectively; and elucidated the catalytic mechanisms using a combination of molecular dynamics (MD) simulations and enzymatic analyses. Full-length RfxMCR was a homodimer of two cross-interlocked subunits, each containing four tandemly arranged short-chain dehydrogenase/reductase (SDR) domains. Only the catalytic domains SDR1 and SDR3 incorporated additional secondary structures that changed with NADP⁺-MSA binding. The substrate, malonyl-CoA, was immobilized in the substrate binding pocket of SDR3 through coordination with Arg1164 and Arg799 of SDR4 and the extra domain (ED), respectively. Malonyl-CoA was successively reduced through protonation by the Tyr743-Arg746 pair in SDR3 and the catalytic triad (Thr165-Tyr178-Lys182) in SDR1 after nucleophilic attack from NADPH hydrides.

Full-length malonyl-CoA reductase (MCR) structure as determined from cryo-electron microscopy (EM) of R. castenholzii malonyl-CoA reductase

Molecular dynamics (MD) simulation and binding free energy calculations of the full-length R. castenholzii malonyl-CoA reductase

文章链接：https://journals.asm.org/doi/10.1128/mbio.03233-22