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 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, Rhodococcus opacus 1CP and Zavarzinia compransoris Z-1155, heterologously overproduced in the cell membrane of 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. IMPORTANCE: Styrene oxide isomerase (SOI) catalyzes the isomerization of styrene oxide into phenylacetaldehyde in the side chain oxygenation of the styrene degradation pathway. Despite performing a key role in this pathway, the biology and biochemistry of this enzyme are poorly understood, particularly its catalytic mechanism, membrane localization, and structure-function relationships. SOIs from Rhodococcus opacus and Zavarzinia compransoris were produced as SUMO/sfGFP/mCherry fusion proteins. We successfully achieved the overproduction and performed site-directed mutagenesis to understand the catalytic mechanism, performed whole-cell assays, used fluorescent microscopy to assess the membrane localization, and constructed terminal truncations to assess structure-function relationships. The site-directed mutagenesis revealed histidine-57 as the axial ligand for heme. The fluorescence microscopy of sfGFP-fusion showed that SOI is a membrane-bound protein with both termini localized in the cytosol. The difference in activity of differently tagged SOI and truncation of the terminal extension showed that the termini might facilitate proper substrate channelling.

Membrane-bound styrene oxide isomerase (SOI) catalyses the Meinwald rearrangement—a Lewis-acid-catalysed isomerization of an epoxide to a carbonyl compound—and has been used in single and cascade reactions. However, the structural information that explains its reaction mechanism has remained elusive. Here we determine cryo-electron microscopy (cryo-EM) structures of SOI bound to a single-domain antibody with and without the competitive inhibitor benzylamine, and elucidate the catalytic mechanism using electron paramagnetic resonance spectroscopy, functional assays, biophysical methods and docking experiments. We find ferric haem b bound at the subunit interface of the trimeric enzyme through H58, where Fe(III) acts as the Lewis acid by binding to the epoxide oxygen. Y103 and N64 and a hydrophobic pocket binding the oxygen of the epoxide and the aryl group, respectively, position substrates in a manner that explains the high regio-selectivity and stereo-specificity of SOI. Our findings can support extending the range of epoxide substrates and be used to potentially repurpose SOI for the catalysis of new-to-nature Fe-based chemical reactions. (Figure presented.).