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Publication Abstract from PubMed

The 2-ketopropyl coenzyme M oxidoreductase/carboxylase (2-KPCC) enzyme is the only member of the disulfide oxidoreductase (DSOR) family of enzymes, which are important for reductively cleaving S-S bonds, to have carboxylation activity. 2-KPCC catalyzes the conversion of 2-ketopropyl coenzyme M (2-KPC) to acetoacetate, which is used as a carbon source, in a controlled reaction to exclude protons. A conserved His-Glu motif present in DSORs is key in the protonation step; however, in 2-KPCC the dyad is substituted by Phe-His. Here we propose this difference is important for coupling carboxylation with C-S bond cleavage. We substituted the Phe-His dyad in 2-KPCC to be more DSOR-like, replacing the phenylalanine with histidine (F501H) and the histidine with glutamate (H506E), and solved crystal structures of F501H and the double variant F501H_H506E. We found that F501 protects the enolacetone intermediate from protons and that the F501H variant strongly promotes protonation. We also provided evidence for the involvement of the H506 residue in stabilizing the developing charge during the formation of acetoacetate, which acts as a product inhibitor in the WT but not the H506E variant enzymes. Finally, utilizing a variety of methods to inhibit the catalytic cysteines shared among all DSORs, we determined that the F501H substitution promotes a DSOR-like charge transfer interaction with FAD, eliminating the need for cysteine as an internal base. Taken together, these results indicate that the 2-KPCC dyad is responsible for selectively promoting carboxylation and inhibiting protonation in the formation of acetoacetate.

The unique Phe-His dyad of 2-ketopropyl coenzyme M oxidoreductase/carboxylase selectively promotes carboxylation and S-C bond cleavage.,Prussia GA, Shisler KA, Zadvornyy OA, Streit BR, DuBois JL, Peters JW J Biol Chem. 2021 Jul 12:100961. doi: 10.1016/j.jbc.2021.100961. PMID:34265301^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.