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Structural insights into the increased activity of engineered glycerol dehydratase

  • 박래윤 (Raeyun Park, 아주대학교 일반대학원)

초록/요약

Glycerol dehydratase (GDHt) is a B12-dependent enzyme that catalyzes the conversion of glycerol to 3-hydroxypropionaldehyde (3-HPA), a valuable platform chemical. In this study, we analyzed the crystal structure of a fused GDHt (fGDHt), in which the α- and β- subunits are connected by a flexible linker to improve stability. The overall structure and B12-binding interface of fGDHt were well conserved compared to the non-fused form. To further enhance enzyme performance, we structurally characterized engineered variants (α-A177M, β-L113W, β-M158W, and α-A177M/β-M158W). These mutations reinforced inter-subunit interactions through hydrophobic contacts, aromatic stacking, and coenzyme oxygen tunnel shielding. Notably, the double mutant showed synergistic structural stabilization, suggesting improved cofactor protection and enzyme integrity. These results demonstrate that subunit interface engineering is an effective strategy for improving the stability of multimeric B12-dependent enzymes. The structural insights gained here may support future development of robust biocatalysts for industrial biotransformation of glycerol-based feedstocks.

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목차

1. Introduction 1
2. Materials & Methods 5
2.1 Materials 5
2.2 Apparatus 5
2.3 Methods 6
2.3.1 Cell culture and protein overexpression 6
2.3.2 Purification of fGDHt and its variants 6
2.3.3 Protein crystallization 7
2.3.4 X-ray diffraction and data collection 8
2.3.5 Structural determination and analysis 8
3. Result & Discussion 9
3.1 Solubility test of fGDHt 9
3.2 Purification of fGDHt and its variants 11
3.3 Crystallization of fGDHt and its variants 15
3.4 Crystal data collection and refinement 17
3.5 Crstal structure of fGDHt and its variants 20
3.5.1 Overall structure of fGDHt and comparison with non-fused GDHt 20
3.5.2 Structural analysis of fGDHt variants 22
4. Conclusion 26
5. Reference 27

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