Poster Presentation 21st International Conference on Biological Inorganic Chemistry 2025

Characterization of a key enzyme in the reductive glycine pathway (#531)

Joseph B Solomon 1 , Andreas M Kueffner 1 , Tobias Krug 1 , Frederik V Schmidt 1 , Johannes G Rebelein 1 , Tobias J Erb 1
  1. Max Planck Institute for Terrestrial Microbiology, Marburg, HESSE, Germany

 

Pyruvate:ferredoxin oxidoreductase (PFOR) is a thiamine pyrophosphate (TPP) dependent FeS enzyme that reversibly catalyzes the conversion of acetyl-CoA and CO2 to pyruvate.1,2 The oxidative decarboxylation of pyruvate by PFOR is used in catabolic pathways to generate low-potential electrons (E0' = -540 mV) that reduce ferredoxins or flavodoxins.3 The reduced ferredoxins or flavodoxins can be used as electron donors for challenging chemical transformations, such as the reduction of N2 by nitrogenase.4 However, in many microbes PFOR is used in anabolic pathways to fix CO2.5,6

Recently, Desulfovibrio desulfuricans (Dd) was shown to grow autotrophically despite lacking the genes required for any of the previously known natural CO2-fixing pathways.7 Based on subsequent multiomic analyses, the 7th naturally occurring CO2-fixing pathway, the reductive glycine pathway (rGlyP), was proposed.  The rGlyP relies on DdPFOR as a CO2-fixing module, but this enzyme was neither isolated nor characterized. Thus, the ability of this enzyme to fix CO2 has yet to be directly demonstrated in the literature. 

In this work, we demonstrate that DdPFOR catalyzes the reductive carboxylation of acetyl-CoA to pyruvate. DdPFOR and its putative electron transfer partner are biochemically characterized and the structure of DdPFOR is determined. Subsequent analyses compare DdPFOR to its well-characterized and closely related homolog, Desulfovibrio africanus PFOR.

  1. Menon S, Ragsdale SW. (1997) Mechanism of the Clostridium Thermoaceticum Pyruvate: Ferredoxin Oxidoreductase: Evidence for the Common Catalytic Intermediacy of the Hydroxyethylthiamine Pyropyrosphate Radical. Biochemistry, 36 (28), 8484–8494.
  2. Thauer RK, Käufer B, Scherer P. (1975) The Active Species of “CO2” Utilized in Ferredoxin-Linked Carboxylation Reactions. Arch. Microbiol., 104 (1), 237–240.
  3. Furdui C, Ragsdale SW. (2000) The Role of Pyruvate Ferredoxin Oxidoreductase in Pyruvate Synthesis during Autotrophic Growth by the Wood-Ljungdahl Pathway. J. Biol. Chem. 275 (37), 28494–28499.
  4. Wahl RC, Orme-Johnson WH. (1987) Clostridial Pyruvate Oxidoreductase and the Pyruvate-Oxidizing Enzyme Specific to Nitrogen Fixation in Klebsiella Pneumoniae Are Similar Enzymes. J. Biol. Chem. 262 (22), 10489–10496.
  5. Yoon, KS, Hille R, Hemann C, Tabita FR. (1999) Rubredoxin from the Green Sulfur Bacterium Chlorobium Tepidum Functions as an Electron Acceptor for Pyruvate Ferredoxin Oxidoreductase. J. Biol. Chem. 274 (42), 29772–29778.
  6. Tersteegen A, Linder D, Thauer RK, Hedderich R. (1997) Structures and Functions of Four Anabolic 2-Oxoacid Oxidoreductases in Methanobacterium Thermoautotrophicum. Eur. J. Biochem. 244 (3), 862–868.
  7. Sánchez-Andrea I, Guedes IA, Hornung B, Boeren S, Lawson CE, Sousa DZ, Bar-Even A, Claassens NJ, Stams AJM. (2020) The Reductive Glycine Pathway Allows Autotrophic Growth of Desulfovibrio Desulfuricans. Nat. Commun. 11 (1), 1–12.