Mechanistic Insight into Peptidyl‐Cysteine Oxidation by the Copper‐Dependent Formylglycine‐Generating Enzyme

Abstract

The copper-dependent formylglycine-generating enzyme (FGE) catalyzes the oxygen-dependent oxidation of specific peptidyl-cysteine residues to formylglycine. Our QM/MM calculations provide a very likely mechanism for this transformation. The reaction starts with dioxygen binding to the tris-thiolate Cu I center to form a triplet Cu II -superoxide complex. The rate-determining hydrogen atom abstraction involves a triplet-singlet crossing to form a Cu II À OOH species that couples with the substrate radical, leading to a Cu Ialkylperoxo intermediate. This is accompanied by proton transfer from the hydroperoxide to the S atom of the substrate via a nearby water molecule. The subsequent OÀ O bond cleavage is coupled with the CÀ S bond breaking that generates the formylglycine and a Cu IIoxyl complex. Moreover, our results suggest that the aldehyde oxygen of the final product originates from O 2 , which will be useful for future experimental work.

Ligand–Copper(I) Primary O2-Adducts: Design, Characterization, and Biological Significance of Cupric–Superoxides

“…Lytic polysaccharide monooxygenases (LPMOs) and particulate -methane monooxygenase ( p MMO), both possessing a “histidine brace” (imidazolyl and chelating amine terminus), effect polysaccharide oxidative breakdown or methane to methanol conversion, respectively; their (bio)­chemistries are critical in energy conversion. The formylglycine generating enzyme (FGE) features a copper­(I) bis-cysteinate which when further ligated by a substrate cysteine reacts with O 2 , giving a Cu II –superoxide which attacks the methylene group via HAA; further steps lead to products C α -formylglycine and H 2 S­(g).…”

New Insights into the Plutella xylostella Detoxifying Enzymes: Sequence Evolution, Structural Similarity, Functional Diversity, and Application Prospects of Glucosinolate Sulfatases

“…The formylation reaction is basic and efficient because the whole process only requires reducing agents and molecular oxygen rather than cofactors or metal ions. ,, However, the latest study reveals the specific role played by copper in the Sumf1-catalyzed oxidation reaction. Based on the computational data of hybrid quantum mechanics/molecular mechanics (QM/MM), Cu­(II)-OOH, covering a hydrogen atom abstraction from the substrate and environmental O 2 , continues to form Cu­(I)-alkylperoxo intermediate, followed by an O–O bond cleavage leading to a FGly . Although no crystal structures of PxSumf1s bound to peptide substrates have been solved, PxSumf1s showed high structural similarity with HsSumf1, leaving the ligand-root-mean-square deviation (RMSD) of PxSumf1a or PxSumf1b between HsSumf1 at 0.163 and 0.186, respectively (Figure A).…”

“…Based on the computational data of hybrid quantum mechanics/molecular mechanics (QM/MM), Cu(II)-OOH, covering a hydrogen atom abstraction from the substrate and environmental O 2 , continues to form Cu(I)-alkylperoxo intermediate, followed by an O−O bond cleavage leading to a FGly. 57 Although no crystal structures of PxSumf1s bound to peptide substrates have been solved, PxSumf1s showed high structural similarity with HsSumf1, leaving the ligand-root-mean-square deviation (RMSD) of PxSumf1a or PxSumf1b between HsSumf1 at 0.163 and 0.186, respectively (Figure 2A). The comparison of HsSumf1, PxSumf1a, and PxSumf1b reveals three distinct areas, with significant differences occurring in areas A1 and A3 and fewer in the catalytic pocket in area A2 (Figure 2A).…”

Promiscuous Enzymes for Residue‐Specific Peptide and Protein Late‐Stage Functionalization

“…[72,73] Aerobic FGEs catalyze the conversion of the thiol side chain of cysteine to an electrophilic aldehyde-bearing group via copper and oxygendependent catalysis (Figure 3B). [74][75][76] Their native function is to activate type-I sulfatases via oxidation and subsequent hydrolysis and their deficiency is associated with several diseases. [77,78] Different bacterial FGEs were found to be promiscuous in recognizing their Cys-containing peptide sequence and modified protein with the tag Cxxxx.…”