Adriano Alves Furtado

Snake-venom phospholipases A ₂ (PLA ₂ s) are small, structurally conserved enzymes that contribute significantly to the pathophysiology of envenomation. Here, we report the purification and crystal structure of an Asp49-PLA ₂ isolated from the venom of Lachesis muta , a pit viper from the Peruvian Amazon. The enzyme was purified using ion-exchange and size-exclusion chromatography and exhibited phospholipase activity in a dose- and time-dependent egg-yolk degradation assay. Pure protein crystals were obtained in space group P 6 ₂ 22 and diffracted to 2.36 Å resolution, with two molecules in the asymmetric unit. The structure reveals the canonical fold of catalytically active group II PLA ₂ s, with a bound Ca ²⁺ ion and a MES molecule in the active site of one monomer. Seven disulfide bonds stabilize the structure, although one bridge typically associated with the β-hairpin is absent and is replaced by a salt bridge as in other viperid PLA ₂ s. PISA analysis suggests a potential tetrameric assembly composed of two AB dimers generating an interface between two A subunits ( A – A ′). Electrostatic surface mapping reveals a notable positively charged channel at the A – A ′ interface, like that seen for a basic PLA ₂ homodimer from Crotalus durissus terrificus in which the two active sites lie accessible to the membrane. This study presents the first structural and enzymatic analysis of an Asp49-PLA ₂ from L. muta and provides insights into its oligomeric assembly, electrostatic landscape and potential adaptations relevant to its role in venom toxicity.

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.