Enolase is a glycolytic and gluconeogenic enzyme also found on the surface of several eukaryotic and prokaryotic cells where it acts as plasminogen binding protein. Leishmania mexicana, one of the causative agents of Leishmaniasis, binds plasminogen and, in this parasite, enolase has been previously found associated with the external face of the plasma membrane. In this work, we show that the purified recombinant enolase has plasminogen binding activity indicating that, at the surface of the parasite, the protein may function as one of the plasminogen receptors. An internal motif (249)AYDAERKMY(257), similar to the nine amino-acid internal plasminogen-binding motif in Streptococcus pneumoniae enolase, is responsible for plasminogen interaction with the parasite enolase. Anti-enolase antibodies inhibited up to 60% of plasminogen binding on live parasites indicating that enolase act as a plasminogen receptor on the parasite. The fact that enolase acts as a possible plasminogen receptor in vivo makes this protein a promising target for therapy.

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Glycolysis and glyconeogenesis play crucial roles in the ATP supply and synthesis of glycoconjugates, important for the viability and virulence, respectively, of the human-pathogenic stages of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. These pathways are, therefore, candidate targets for antiparasite drugs. The glycolytic/gluconeogenic enzyme enolase is generally highly conserved, with similar overall fold and identical catalytic residues in all organisms. Nonetheless, potentially important differences exist between the trypanosomatid and host enzymes, with three unique, reactive residues close to the active site of the former that might be exploited for the development of new drugs. In addition, enolase is found both in the secretome and in association with the surface of Leishmania spp. where it probably functions as plasminogen receptor, playing a role in the parasite's invasiveness and virulence, a function possibly also present in the other trypanosomatids. This location and possible function of enolase offer additional perspectives for both drug discovery and vaccination.

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Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases. We successfully express, purify, and assay 51 enzymes from seven distinct phylogenetic groups; observing PET hydrolysis activity on amorphous PET film from 37 enzymes in reactions spanning pH from 4.5–9.0 and temperatures from 30–70 °C. We conduct PET hydrolysis time-course reactions with the best-performing enzymes, where we observe differences in substrate selectivity as function of PET morphology. We employed X-ray crystallography and AlphaFold to examine the enzyme architectures of all 74 candidates, revealing protein folds and accessory domains not previously associated with PET deconstruction. Overall, this study expands the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction.