New design of a chimeric enzyme based on vanadium-dependent bromoperoxidase and glucose oxidase to highlight the microbicidal properties of the reaction products: HOBr and HOCl.

Abstract

Vanadium-dependent haloperoxidases are enzymes found in bacteria, fungi and red or brown macroalgae. They are used as a defense system for the latter by producing bromoform. The intermediate product, HOBr or HOCl, is also a microbicidal compound that can be exploited in many domains as medical tools or disinfectant sprays for example. We show that the mutation identified in Ohshiro's work, which alters substrate specificity toward chloride, is transferable to the homologous enzyme from Chondrus crispus. We then applied genetic and enzymatic engineering to design a chimera with glucose oxidase, thereby enhancing antimicrobial properties by providing a local source of H2O2. We used the SpyTag/SpyCatcher technology to form the chimera and obtained homogenous objects, indicating that one oligomeric form is favored. We demonstrated the release of HOBr and HOCl thanks to NADH that reacts spontaneously with it outside the active site of the enzyme. We also measured the steady-state kinetic parameters of the wild-type or modified enzymes. The formation of the chimera increased the specificity of ccVHPO1 inside the chimera, towards KBr or H2O2, compared to the enzyme alone with the SpyCatcher. Finally, we showed a significant increase in the microbicidal effect between a coupled enzymatic system with the glucose oxidase (the two enzymes are free in solution) compared to the chimera system, which is completely bactericidal at concentrations around 20nM.