Heme enzymes can perform a wide range of reactions in biological systems, often controlled by the heme surrounding amino acids or in conjunction with redox partners. Recently, we resolved the cryo-EM structure of styrene oxide isomerase, a transmembrane protein that catalyzes the isomerization of epoxides into carbonyl compounds. We discovered that heme acts as the cofactor and catalytic center, with tyrosine serving as the key residue in catalysis. While it is evident that tyrosine coordinates the substrate in the active site, the catalytic mechanism is not fully established yet. In this work, we advanced the investigation into a homologous enzyme to explore whether tyrosine plays a conserved role in catalysis. Site-directed mutagenesis of this tyrosine (Y131) to Ala, His, Ser, and Phe demonstrated a significant effect on the activity and kinetic parameters. The pH-dependent activity assay and inhibition of the heme site with carbon monoxide illustrated both ferric heme and tyrosine to be crucial for catalysis. The NMR-based enzyme kinetics suggested that heme b acts as a Lewis-acid in ring opening and Y131 facilitates the trans-methyl/hydride shift to perform the Meinwald-rearrangement reaction. Furthermore, our findings indicate that active heme can be utilized for various reactions, functioning as peroxidase with a turnover number (k_cat) of 2 s^–1and as a peroxygenase with a total yield of 10% phenylacetaldehyde. Although its peroxidase activity is ca. 1000-fold less efficient compared to dye decolorizing peroxidase DyP, the multifunctionality of ZcSOI makes it an intriguing enzyme for application studies. This work deepens our understanding of the SOI mechanism and also reports on the less efficient peroxidase and peroxygenase activity. This, in turn makes SOI a promising candidate for protein engineering to apply in the field of biotechnology, such as improving the epoxidation and isomerization of styrene to produce phenylacetaldehyde by a single enzyme.

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.

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.).