Fig. 5: The study on the nucleophilic catalytic residue of C26.

a 2-Cyclopentenone labeling of GkOYE.8 measured by ESI MS. b 2-Cyclopentenone labeling of GkOYE.8^C26A measured by ESI MS. Both (a) and (b) were labeled at 5 mM 2-cyclopentenone for 2 h. 2-cyclopentenone labels specifically on the GkOYE.8 (peak position marked by red circle) but not on the GkOYE.8^C26A. c The proposed mechanism and Gibbs free energy profile of MBH reaction without enzyme calculated by DFT. The putative mechanism of MBH reaction consists of 5 steps^57,58. The step 1: Michael addition. HPO₄^2- as a nucleophile attacks substrate 1⁵⁹. The step 2: Aldol reaction. The step 3 and 4: Proton transfer. The step 5: Elimination. d The proposed mechanism and Gibbs free energy profile of MBH reaction with a theozyme model calculated by DFT. The theozyme model includes substrate 1, substrate 2, water, methanethiol (substituting for residue C26) and acetic acid (substituting for residue E59). DFT-computed Gibbs free energies (in kcal/mol) at the CPCM(water)/B3LYP-D3/6-311 + + G(2d,p)//CPCM(water)/B3LYP-D3/6-31 + G(d) level of theory and transition-state structures (carbon: gray, hydrogen: white, oxygen: red, nitrogen: blue, sulfur: yellow, phosphorus: orange and distances are shown in Å). e The role of E59 site analyzed by the MD simulation. f Exploration the effect of pH on MBH reaction. n = 3 independent biological experiments. Data are presented as mean values ± SD. Source data are provided as a Source Data file.