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

Abstract

Enzyme cascade reactions hold transformative potential for sugar nucleotide biosynthesis, aligning with green chemistry principles by minimizing solvent waste and purification steps. However, their potential is often compromised by low catalytic efficiency due to inefficient intermediate utilization and instability of individual enzymes. 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 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 acts as a sustainable nano-reactor, not only stabilizing the dual-enzyme conjugate conformation but also elevating local substrate concentrations (ATP, UTP, and GlcNAc) via synergistic electrostatic and van der Waals interactions, thereby enhancing reaction kinetics and resource efficiency. Using stimulated Raman scattering (SRS) microscopy, we directly visualized the spatial confinement and rapid consumption of the intermediate substrate GlcNAc-1-P on the nanocomposite surface, demonstrating an engineered substrate channeling-like effect, a common mechanism in native metabolon complexes that boosts cascade efficiency. The resulting nanocomposite exhibits a 4.4-fold higher activity than free enzymes and superior stability across varying temperatures and pH conditions, and retains approximately 60% of its initial activity after five reuse cycles. This work establishes a generalizable and robust strategy for constructing metal–organic framework (MOF)–enzyme complexes with broad applicability in high-value sugar nucleotide biosynthesis and other complex bioconversion processes requiring metabolic flux control.