Structures of BlEst2 from Bacillus licheniformis in its propeptide and mature forms reveal autoinhibitory effects of the C-terminal domain.

ABSTRACT

Carboxylesterases comprise a major class of α/ß-fold hydrolases responsible for the cleavage and formation of ester bonds. Found ubiquitously in nature, these enzymes are crucial for the metabolism of both endogenous and exogenous carboxyl esters in animals, plants and microorganisms. Beyond their essential physiological roles, carboxylesterases stand out as one of the important classes of biocatalysts for biotechnology. BlEst2, an enzyme previously classified as Bacillus licheniformis esterase, remains largely uncharacterized. In the present study, we elucidate the structural biology, molecular dynamics and biochemical features of BlEst2. Our findings reveal a canonical α/ß-hydrolase fold similar to the ESTHER block L of lipases, further augmented by two additional accessory C-terminal domains. Notably, the catalytic domain demonstrates two insertions, which occupy conserved locations in α/ß-hydrolase proteins and commonly form the lid domain in lipase structures. Intriguingly, our in vitro cleavage of C-terminal domains revealed the structure of the active form of BlEst2. Upon activation, BlEst2 showed a markedly elevated hydrolytic activity. This observation implies that the intramolecular C-terminal domain serves as a regulatory intramolecular inhibitor. Interestingly, despite exhibiting esterase-like activity, BlEst2 structural characteristics align more closely with lipases. This suggests that BlEst2 could potentially represent a previously unrecognized subgroup within the realm of carboxyl ester hydrolases.