Enzymes are relatively large protein molecules (>10,000 Da) that accelerate extremely important cellular chemical reactions with high substrate specificity (chemo-, regio- and stereo-), reaction rates, and yields under mild reaction conditions.1 Precisely determined by the primary amino acids sequence, the unique three-dimensional folded structure of an enzyme is crucial for its appropriate function. Moreover, enzymes bear great promise as environmentally friendly biocatalytic alternatives in numerous research and industrial applications, including food, health, cosmetics, agriculture, environment, energy and applied enzymology, an important branch of industrial biotechnology.2 Among the pool of enzymes, carbonic anhydrases (CA) are one of the most efficient biocatalysts, facilitating the hydration of CO2 and ester hydrolysis, thus potentially bearing global biotechnological implications.3 Despite their significant advantages, the intrinsic drawbacks of enzymes, including low operational stability (thermal and long-term viability), the sensitivity of catalytic activity to environmental conditions (pH and solvent), high costs associated with preparation and purification and difficulties in recovery and recycling, dramatically hinder their practical utilization. Therefore, development of a simple artificial analog that can mimic the active site of natural CA and provide high catalyst stability and low production costs is highly desirable for biotechnological and industrial applications.
The catalytic center of CA is composed of a Zn(II) ion coordinated by three histidine residues extending from adjacent antiparallel β-strand protein scaffolds. This structure has inspired researchers to develop many peptide β-sheet supramolecular CA mimetics with high catalytic efficiencies comparable to the natural enzyme per weight (Figure 1).4,5 However, in spite the success of initial peptide-based studies of amyloid-associated CA mimics, single amino acid assemblies have so far not been examined for similar biocatalytic applications. Based on our extensive research experience studying phenylalanine (F) and its amyloid-like highly-ordered self-assembly characteristics,6 we set out to explore the possible CA-like catalytic activity of F assemblies in the presence of zinc ions as a co-factor. To our surprise, F and Zn(II) spontaneously coordinated to form long needle-like crystals with a layered supramolecular amyloid-like ordered crystal lattice (F-Zn(II)). More importantly, the F-Zn(II) single crystals compellingly exhibited two CA catalytic activities, both as esterase and in CO2 hydration. Remarkably, among the reported artificial biomolecular hydrolases, F-Zn(II) displayed the lowest molecular weight and highest catalytic efficiency (Figure 1), in addition to its reusability, thermal stability, substrate specificity, stereoselectivity, and rapid catalytic CO2 hydration ability. We performed a detailed experimental and computational structural analysis of the active species and found the extraordinary catalytic activity was related both to the unique amyloid-like supramolecular structural framework and the dense ordered array of zinc ions on the surface of the crystals. Thus, this study provides a novel minimalistic de novo design of an enzyme mimetic, allowing to further expand the scope of metabolite assemblies, and sets a new stage for the discovery of robust biocatalysts to address the present and future biotechnological and industrial challenges.
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