Bifunctional chimeras of myeloperoxidase and glucose oxidase. Antimicrobial, topological and enzymatic properties

Abstract

Enhancing the local substrate concentration is a crucial strategy in nature for facilitating the proximity of two enzymes. The substrate of the first enzyme is transformed into a by-product that travels to the active site of the second enzyme without external diffusion, then transformed into a product and eventually expelled from the complex. In an effort to optimize the antimicrobial properties of myeloperoxidase from Rhodopirellula baltica (RbMPO), we created a library of fused chimeras between a glucose oxidase (GOx) and RbMPO so that H₂O₂ could be continuously perfused in the vicinity RbMPO, enabling the production of HOCl or HOSCN, well-known antimicrobial agents. The enzymes were characterized biochemically, enzymatically, and physically using low-resolution techniques such as AFM, SAXS, and cryofracture. SAXS experiments revealed that the chimeras were properly folded and existed in different oligomeric states. The kinetic parameters of the chimeras were determined and used for classification, revealing that all chimeras exhibited varying levels of activity and were microbicidal. The mixture of different oligomeric states of LEGGEAEA displayed both activity and microbicidal properties. AFM was used to visualize the chimeras in different oligomeric states, with their overall shapes ranging from round, oblong, to hooked, depending on the linker used.