Structural and catalytic alteration of sarcosine oxidase through reconstruction with coenzyme-like ligands

A sarcosine oxidase (SOX) gene from Bacillus sp. (AY626822.2) was expressed in Escherichia coli BL21 (DE3) in the form of inclusion bodies. A 3D model of SOX was then built and refined, and molecular docking was used to investigate the interactions between SOX and natural or coenzyme-like ligands, including flavin adenine dinucleotide (FAD); flavin mononucleotide (FMN); riboflavin; isoalloxazine; 7-methyl-8-chloro-10-(1′-d-ribityl) isoalloxazine (7-M-8-C); 7-bromo-8-methyl-10-(1′-d-ribityl) isoalloxazine (7-B-8-M); 7-methyl-8-bromo-10-(1′-d-ribityl) isoalloxazine (7-M-8-B); 7-chloro-8-ethyl-10-(1′-d-ribityl) isoalloxazine (7-C-8-E); 7,8-diethyl-10-(1′-d-ribityl) isoalloxazine (7,8-D); and 3-methyl-7,8-dimethyl-10-(1′-d-ribityl) isoalloxazine (3-M-7,8-D). Unfolded SOX was extracted from inclusion bodies, and reconstructed with these ligands via a refolding process. The reconstructed enzymes were then subjected to structural and catalytic analysis. After structural simulation, refinement, and molecular docking, all ligands were able to recognize the coenzyme site of SOX. In addition, when the position 7- or 8-site of the compounds was modified, new pi-cation/sigma interactions were formed in the SOX-ligand complex. Fluorescent detection revealed that all the ligands could be successfully reconstructed with unfolded SOX. Circular dichroism (CD) spectra and nano differential scanning calorimetry (DSC) analysis indicated that the loss of phosphoric acid and adeninein natural coenzymes could significantly reduce the α-helix content, transition temperature (T_m), and calorimetric enthalpy (ΔH). In addition, although reconstruction with the position 7- or 8-site modified compounds led to variations in secondary structure, no significant shifts in T_m and ΔH were observed. Furthermore, in the evaluation of catalytic kinetic parameters, when SOX was reconstructed with ligands containing halogen atoms at the 7- or 8-sites, much higher relative specificities in the presence of organic solvents were noted.