Functional characterization and membrane localization of the styrene oxide isomerase from \(\textit {Rhodococcus opacus}\) 1CP and \(\textit {Zavarzinia compransoris}\) Z-1155

Styrene oxide isomerase (SOI) is a part of the styrene degradation enzyme complex, performing the isomerization of toxic intermediate styrene oxide into phenylacetaldehyde. For many years, the enzyme was believed to be cofactor-independent, and hence, the mechanism of this enzyme was proposed to be acid-base catalysis. Recently, the presence of heme was identified and reported in SOI from \(\textit {Pseudomonas sp.}\) VLB120. Alongside, the membrane localization was also postulated since its discovery but lacks experimental proof. In this study, we highlight the localization of SOIs from two bacterial strains, \(\textit {Rhodococcus opacus}\) 1CP and \(\textit {Zavarzinia compransoris}\) Z-1155, heterologously overproduced in the cell membrane of \(\textit {E. coli}\) via sfGFP-tagged fusions. In addition, the site-directed mutagenesis of acidic and basic amino acids in SOI from 1CP also showcased that histidine-57 is the axial ligand to the heme. Electron paramagnetic resonance (EPR) and biocatalytic assays showed arginine-111 possibly coordinating the propionate group of heme. The functional assays of differently tagged sfGFP with and without linkers, and the truncation of the terminal extension of SOI from 1CP and Z-1155, indicate their possible role in proper substrate channeling. It also supports the previously proposed SOI role as a membrane anchor for other enzymes in styrene degradation pathway.