Fig. 3: Enhancing MBH function of GkOYE from the perspective of catalytic mechanism.

a The mechanism of natural reduction reaction catalyzed by OYEs and the putative mechanism of MBH reaction in this study. The mechanism of MBH reaction was inferred based on the reported mechanisms of enzyme catalysis²³ and chemical small molecule catalysis⁵⁶. Nu: The nucleophilic catalytic residue in GkOYE protein. IntD: The covalent intermediates are formed by the nucleophilic catalytic residue Nu with the βC of the double bond in substrate 1 and the aldehyde group C in substrate 2. b Conservation analysis of the residues interacting with FMN in the GkOYE protein. c Color and SDS-PAGE validation for wild-type and mutant protein. Note: 1-7 correspond to different variants of the protein. 1: GkOYE.1、2: GkOYE.2、3: GkOYE.3、4: GkOYE.4、5: GkOYE.5、6: GkOYE.6、7: GkOYE.7. Three times each experiment was repeated independently with similar results. d Comparison of reduction activity between GkOYE and variant GkOYE.7. Columns 1 and 2 represent the reduction activity of GkOYE.7 with and without FMN. Columns 3 and 4 represent the reduction activity of GkOYE with and without FMN. Columns 5-8 represent blank control groups. + indicates presence and - indicates absence. The added components are 5 mM 1, 1 mM NADPH, 1 mM FMN, and 100 μM purified protein in PBS (pH 7.4) with 1% methanol (MeOH) as a co-solvent. e Comparison of catalytic activity of MBH reaction between GkOYE and variant GkOYE.7. The reactions were carried out using 5 mM 1, 1 mM 2, and 100 μM purified protein in PBS (pH 7.4) with 3% methanol (MeOH) as a co-solvent. f Validation of reduction activity for different mutants. g Comparison of catalytic activity for MBH reaction between different mutants. n = 3 independent biological experiments. Data are presented as mean values ± SD. Source data are provided as a Source Data file.