Susana A. Sculaccio

L-Ascorbic acid (AsA, vitamin C) is a pivotal dietary nutrient with multifaceted importance in living organisms. In plants, the Smirnoff-Wheeler (SW) pathway is the primary route for AsA biosynthesis and understanding the mechanistic details behind its component enzymes has implications for plant biology, nutritional science and biotechnology. As part of an initiative to determine the structures of all six core enzymes of the pathway, the present study focusses on three of them from the model system Myrciaria dubia (camu-camu): GDP-D-mannose 3',5'-epimerase (GME), L-galactose dehydrogenase (L-GalDH), and L-galactono-1,4-lactone dehydrogenase (L-GalLDH). We provide insights into substrate and cofactor binding and the conformational changes they induce. The MdGME structure reveals a distorted substrate in the active site, pertinent to the catalytic mechanism. MdL-GalDH shows that the way in which NAD⁺ association affects loop structure over the active site is not conserved when compared with its homologue from spinach. Finally, the structure of MdL-GalLDH is described for the first time. This allows for the rationalization of previously identified residues which play important roles in the active site or in the formation of the covalent bond with the FAD. In conclusion, this study enhances our understanding of AsA biosynthesis in plants and the information provided should prove useful for biotechnological applications.

Several hydrolases have been described to degrade polyethylene terephthalate (PET) at moderate temperatures ranging from 25°C to 40°C. These mesophilic PET hydrolases (PETases) are less efficient in degrading this plastic polymer than their thermophilic homologs and have, therefore, been the subject of many protein engineering campaigns. However, enhancing their enzymatic activity through rational design or directed evolution poses a formidable challenge due to the need for exploring a large number of mutations. Additionally, evaluating the improvements in both activity and stability requires screening numerous variants, either individually or using high‐throughput screening methods. Here, we utilize instead the design of chimeras as a protein engineering strategy to increase the activity and stability of Mors1, an Antarctic PETase active at 25°C. First, we obtained the crystal structure of Mors1 at 1.6 Å resolution, which we used as a scaffold for structure‐ and sequence‐based chimeric design. Then, we designed a Mors1 chimera via loop exchange of a highly divergent active site loop from the thermophilic leaf‐branch compost cutinase (LCC) into the equivalent region in Mors1. After restitution of an active site disulfide bond into this chimera, the enzyme exhibited a shift in optimal temperature for activity to 45°C and an increase in fivefold in PET hydrolysis when compared with wild‐type Mors1 at 25°C. Our results serve as a proof of concept of the utility of chimeric design to further improve the activity and stability of PETases active at moderate temperatures.