Abstract
Lysophospholipase_carboxylesterase (LPCE) has highly conserved homologs in many diverse species ranging from bacteria to humans, as well as substantial biological significance and potential therapeutic implications. However, its biological function and catalytic mechanism remain minimally investigated because of the lack of structural information. Here, we report the crystal structure of a bacterial esterase PE8 belonging to the LPCE family. The crystal structure of PE8 was solved with a high resolution of 1.66 Å. Compared with other homologs in the family, significant differences were observed in the amino acid sequence, three-dimensional structure, and substrate-binding pattern. Residue Arg79 undergoes configuration switching when binding to the substrate and forms a unique wall, leading to a relatively closed cavity in the substrate-binding pocket compared with the relatively more open and longer clefts in other homologs. Moreover, the mutant Met122Ala showed much stronger substrate affinity and higher catalytic efficiency because less steric repulsion acted on the substrates. Taken together, these results showed that, in PE8, Arg79 and Met122 play important roles in substrate binding and the binding pocket shaping, respectively. Our study provides new insight into the catalytic mechanism of LPCE, which may facilitate the development of structure-based therapeutics and other biocatalytic applications.
Highlights
Esterases have important physiological roles and biotechnological applications because they can catalyze the hydrolysis of short-chain ester-containing molecules and produce carboxylates and alcohols[1,2,3]
Multi-angle light scattering (MALS) analysis showed that the molecular weight (MW) of PE8 was 26.9 kDa (±2.4%) (Fig. 1A), consistent with the theoretical MW of 6× His fusion PE8 (25.4 kDa), and revealed that PE8 existed as a monomer in solution
The structural information of P. halotolerans PE8 expands our knowledge of the catalytic mechanisms of the LPCE family and provides new insight into the substrate-binding pattern in this family
Summary
Esterases have important physiological roles and biotechnological applications because they can catalyze the hydrolysis of short-chain ester-containing molecules and produce carboxylates and alcohols[1,2,3]. Esterases belong to the lysophospholipase_carboxylesterase family (the LPCE family)[4] and were previously classified in bacterial family VI by Arpigny and Jaeger[1]. This family includes the smallest carboxylesterase (23–26 kDa) found to date, and bacterial carboxylesterases show high sequence similaritiy with their eukaryotic counterparts (~40%)[1]. LPCE family proteins play significant roles in human diseases. LPCE family enzymes are increasingly pharmaceutically interesting as potential therapeutic targets. The crystal structures of only six LPCE family proteins have been reported, including Rhodobacter sphaeroides RspE12, Pseudomonas aeruginosa PA385913, P. fluorescens esterase II14, F. tularensis FTT25811, human APT115 and human LYPLAL17. We obtained and analyzed the crystal structure of PE8 to gain new insight into the catalytic mechanism of LPCE family enzymes
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