Construction of a multifunctional enzyme complex via the strain-promoted azide-alkyne cycloaddition.

Inspired by the multienzyme complexes occurring in nature, enzymes have been brought together in vitro as well. We report a co-localization strategy milder than nonspecific cross-linking, and free of any scaffold and affinity tags. Using non-natural amino acid incorporation, two heterobifunctional linkers, and the strain-promoted azide-alkyne cycloaddition as conjugation reaction, three metabolic enzymes are linked together in a controlled manner. Conjugate formation was demonstrated by size-exclusion chromatography and gel electrophoresis. The multienzyme complexes were further characterized by native mass spectrometry. It was shown that the complexes catalyzed the three-step biosynthesis of piceid in vitro with comparable kinetic behavior to the uncoupled enzymes. The approach is envisioned to have high potential for various biotechnological applications, in which multiple biocatalysts collaborate at low concentrations, in which diffusion may be limited and/or side-reactions are prone to occur.