Montpellier Supagro, UMR 1208 IATE, - 2 place Viala, 34060 Montpellier cedex 2, France.
Montpellier Supagro, UMR 1208 IATE, - 2 place Viala, 34060 Montpellier cedex 2, France; Ecole Nationale Supérieure de Chimie de Montpellier, 8 rue de l'Ecole Normale, 34296 Montpellier, France.
Biochim Biophys Acta Proteins Proteom. 2017 Sep;1865(9):1105-1113. doi: 10.1016/j.bbapap.2017.06.004. Epub 2017 Jun 13.
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.
假丝酵母属 CpLIP2 同源的脂肪酶/酰基转移酶属于 α/β 水解酶超家族，与南极假丝酵母脂肪酶 A（南极假丝酵母）具有至少 56%的同源性，构成了一个一致的系统发育亚群。脂肪酶/酰基转移酶具有表型特征，即使在水热力学活性非常高的水介质中，也具有很高的酰基转移活性。以前的诱变和进化策略使我们深入了解了关键残基和蛋白质亚结构域在这些酶的反应和底物特异性中的作用。然而，多次突变通常对活性有害，并且确定所有导致功能的残基都很复杂。本研究采用新的互补方法阐明结构决定因素，基于祖先蛋白的复活来了解进化如何导致生物催化剂目前的特性。通过这样做，与现存蛋白质的比较可以确定参与酶专业化的关键残基。通过祖先序列重建，我们生成了一个假定的祖先脂肪酶/酰基转移酶，PaleoLAc。该酶与 CpLIP2 具有高度的同一性，但催化行为不同。PaleoLAc 确定了参与酰基转移能力的假定关键残基，并支持这样一种假设，即在脂肪酶/酰基转移酶家族中，这种特殊性质与活性位点附近的一组残基有关。作为现代脂肪酶/酰基转移酶观察到的催化行为多样性的祖先起源的代表，PaleoLAc 为进一步针对目标专业化进行工程设计提供了有力工具。
