A glimpse into the specialization history of the lipases/acyltransferases family of CpLIP2

- A functional Ancestral protein of CpLIP2 lipase/acyltransferase family was obtained by Ancestral Sequence Reconstruction
- Compared to the lipase/acyltransferase of the family, PaleoLAc displayed lower and less specialized activity
- Considering is low specificity, PaleoLAc is a good starting material to be improved for targeted applications
- Structure/function relationships can be elucidated by comparison of PaleoLAc with extent lipase/acyltransferases

Lipases/acyltransferases homologous to CpLIP2 from Candida parapsilosis belong to the α/β hydrolase superfamily as lipase A from Moesziomyces antarcticus (Candida antarctica), and constitute a consistent phylogenetic subgroup with at least 56% identity. Lipases/acyltransferases share the phenotypic characteristic of a high acyltransfer activity even in aqueous media with very high water thermodynamic activity. Previous mutagenesis and evolution strategies have given insights into the role of key residues and protein subdomains in the reaction and substrate specificities of these enzymes. However, multiple mutations are often deleterious for the activity and the identification of all the residues that historically led to the function is complicated. A new complementary approach to elucidate structural determinant was conducted in this study, based on the resurrection of ancestral proteins to understand how the evolution led to the present properties of the biocatalysts. By doing so, the comparison with the extant proteins can lead to the identification of key residues involved in the enzymes' specialization. Using Ancestral Sequence Reconstruction, we have generated a putative ancestral lipases/acyltransferases, PaleoLAc. This enzyme shares a high level of identity with CpLIP2 but has a different catalytic behavior. PaleoLAc allowed the identification of putative key residues involved in acyltransfer ability and supports the hypothesis that this exceptional property within the lipases/acyltransferases family is linked to a cluster of residues in the vicinity of the active site. As a representative of the ancestral origin of the diversity of the catalytic behaviors observed in modern lipases/acyltransferases, PaleoLAc constitutes a powerful tool for further engineering toward targeted specialization.