Presentation Information
[2ASPR-04]Engineering Programmable Protein Crystalline Inclusions for ATP Regeneration
○JIAXIN TIAN1 (1. School of Life Science and Technology. Institute of Science Tokyo (Japan))
Keywords:
Protein crystal,Crystalline inclusion protein A (CipA),enzyme immobilization,NAD(P)(H) cycle,ATP regeneration
«Purpose» Protein crystals are highly ordered solid assemblies that exhibit remarkable robustness and intrinsic porosity. Therefore, they are considered as promising platforms for solid-state catalytic materials1. Through amino acid engineering, both small molecules and proteins can be immobilized within crystalline lattices to generate functional hybrid materials2. Crystalline inclusion protein A (CipA)3 forms stable intracellular crystalline inclusions via tetrameric assembly and has been widely utilized as a genetic scaffold for organizing exogenous proteins into solid crystalline structures within living cells.
In this study, we exploit CipA as programmable scaffolds to immobilize multiple ATP-regenerating enzymes, including formate dehydrogenase (FDH), NAD˜ kinase (NADK), polyphosphate-dependent NAD˜ kinase (PPNK), and NADPH oxidase (TPNOX)4. By separately generating enzyme-immobilized crystalline inclusions and subsequently combining these catalytic crystal modules, we establish a modular and spatially organized platform for ATP regeneration. This approach demonstrates the potential of protein crystals as solid-state biocatalytic systems for cofactor recycling and energy regeneration.
«Method»
Four ATP-regenerating enzymes were genetically fused to the N- or C-terminus of CipA, forming crystalline inclusions in E. coli, which were isolated by sonication. Crystal formation and morphology were analyzed by SDS-PAGE, SEM, and SAXS. Enzymatic activity was assessed by measuring absorbance at 340 nm, and ATP production was quantified using a luciferase assay.
«Results»
We successfully expressed four enzymes fused to CipA. The enzymatic activity varied depending on whether the enzymes were fused to the N- or C-terminus of CipA.
«Conclusion»
In conclusion, we engineered CipA-based crystalline inclusions as immobilization scaffolds for ATP-regenerating enzymes. The results demonstrated that the enzyme–crystal modules retain catalytic activity and function as modular solid-state biocatalytic platforms. The system performance depends on fusion orientation and structural constraints, substrate diffusion within the porous lattice.
Reference:1). L. Zhang, J. B. Bailey, R. H. Subramanian, A. Groisman, F. A. Tezcan, Nature. 2018, 557, 86. 2). K. Han, Y. Na, L. Zhang, F. A. Tezcan, J. Am. Chem. Soc. 2022, 144, 10139. 3). S. B. Bintrim, J. C. Ensign, J. Bacteriol. 1998, 180, 1261. 4). E. Willett and S. Banta, ACS Synth. Biol.2023, 12, 2118.
In this study, we exploit CipA as programmable scaffolds to immobilize multiple ATP-regenerating enzymes, including formate dehydrogenase (FDH), NAD˜ kinase (NADK), polyphosphate-dependent NAD˜ kinase (PPNK), and NADPH oxidase (TPNOX)4. By separately generating enzyme-immobilized crystalline inclusions and subsequently combining these catalytic crystal modules, we establish a modular and spatially organized platform for ATP regeneration. This approach demonstrates the potential of protein crystals as solid-state biocatalytic systems for cofactor recycling and energy regeneration.
«Method»
Four ATP-regenerating enzymes were genetically fused to the N- or C-terminus of CipA, forming crystalline inclusions in E. coli, which were isolated by sonication. Crystal formation and morphology were analyzed by SDS-PAGE, SEM, and SAXS. Enzymatic activity was assessed by measuring absorbance at 340 nm, and ATP production was quantified using a luciferase assay.
«Results»
We successfully expressed four enzymes fused to CipA. The enzymatic activity varied depending on whether the enzymes were fused to the N- or C-terminus of CipA.
«Conclusion»
In conclusion, we engineered CipA-based crystalline inclusions as immobilization scaffolds for ATP-regenerating enzymes. The results demonstrated that the enzyme–crystal modules retain catalytic activity and function as modular solid-state biocatalytic platforms. The system performance depends on fusion orientation and structural constraints, substrate diffusion within the porous lattice.
Reference:1). L. Zhang, J. B. Bailey, R. H. Subramanian, A. Groisman, F. A. Tezcan, Nature. 2018, 557, 86. 2). K. Han, Y. Na, L. Zhang, F. A. Tezcan, J. Am. Chem. Soc. 2022, 144, 10139. 3). S. B. Bintrim, J. C. Ensign, J. Bacteriol. 1998, 180, 1261. 4). E. Willett and S. Banta, ACS Synth. Biol.2023, 12, 2118.
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