Spatial organization of an enzyme cascade in Ni-ZIF-8 framework for efficient sugar nucleotide synthesis

Abstract

Enzyme cascade reactions hold transformative potential for sugar nucleotide biosynthesis, yet are often limited by low catalytic efficiency due to inefficient intermediate utilization and instability of individual enzyme. Capitalizing on the coordinatively unsaturated Ni²⁺ sites and the hydrophilic nature of Ni-doped zeolitic imidazolate framework-8 (Ni-ZIF-8), we engineered a highly active nanocomposite by incorporating of a hexahistidine-tagged dual-enzyme conjugate of N-acetylhexosamine 1-kinase (BlNahK) and N-acetylglucosamine 1-phosphate uridylyltransferase (PmGlmU) (His₆-BlNahK-PmGlmU-His₆) for efficient synthesis of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc)—a pivotal sugar nucleotide. The Ni-ZIF-8 scaffold not only stabilizes the dual-enzyme conjugate conformation but also elevates local substrate concentrations (ATP, UTP, GlcNAc) through synergistic electrostatic and van der waals interactions. Using stimulated Raman scattering (SRS) microscopy, we directly visualize the spatial confinement and rapid consumption of the intermediate substrate GlcNAc-1-P on the nanocomposite surface, demonstrating a substrate channeling-like effect, a common mechanism in native metabolon complex, that boosts cascade efficiency. The nanocomposite exhibits a 4.4-fold higher activity than free enzymes, displays superior stability under varying temperatures and pH conditions, and retains approximately 60% of its initial activity after five reuse cycles. This study presents a generalizable approach for constructing robust Metal–organic frameworks (MOFs)-enzyme complex with broad applicability in sugar nucleotide biosynthesis and other complex bioconversion processes requiring metabolic flux control.