Fig. 2: The dual roles of YcfA in adenylation and L-Cys adduct formation.

a A two-layered heptameric architecture of YcfA, with each monomer distinctly colored. b The catalytic cavities of adjacent monomers across the two heptameric layers. c ATP binding mode in the YcfA-ATP complex structure. Interaction networks formed between ATP and surrounding residues of YcfA, with hydrogen bonds represented as blue sticks. d Adenylation activity assay of YcfA mutants targeting ATP binding. The WT YcfA is regarded as possessing 100% enzyme activity. The mean is displayed ±SD for n = 3 biological replicates. e GTP binding mode in the YcfA-GTP complex structure. The polar interactions formed between GTP and surrounding residues are represented as blue sticks, and the π-π stacking interaction is shown as yellow stick. f Adenylation activity assay of YcfA mutants targeting GTP binding. The WT YcfA is regarded as possessing 100% enzyme activity. The mean is displayed ±SD for n = 3 biological replicates. g Surface representation of YcfA^D19A-ATP-GTP complex. ATP and GTP are shown as sticks, Mg²⁺ is shown as green sphere. h The cooperative binding of ATP and GTP in the complex structure of YcfA^D19A-ATP-GTP. Interaction networks in the active site involving ATP, GTP, Mg²⁺, and surrounding residues are presented, with polar contacts represented as blue sticks. i Docking simulations of YcfA with adenylated-GTP and L-Cys. The surrounding residues of L-Cys binding are presented, with potential polar contacts represented as blue sticks. j Mutagenesis analysis of the L-Cys binding site shows YcfA’s relative activity in forming the 6-Cys-GTP S-adduct. The WT YcfA is regarded as possessing 100% enzyme activity. The mean is displayed ±SD for n = 3 biological replicates. k Proposed catalytic mechanism of YcfA. Key residues involved in adenylation and L-Cys nucleophilic addition are indicated. Source data are provided as a Source Data file.