The genome of the lactic acid bacterium Lactobacillus plantarum WCFS1 reveals the presence of a rich repertoire of esterases and lipases highlighting their important role in cellular metabolism. Among them is the carboxylesterase LpEst1 a bacterial enzyme related to the mammalian hormone-sensitive lipase, which is known to play a central role in energy homeostasis. In this study, the crystal structure of LpEst1 has been determined at 2.05 A resolution; it exhibits an alphabeta-hydrolase fold, consisting of a central beta-sheet surrounded by alpha-helices, endowed with novel topological features. The structure reveals a dimeric assembly not comparable with any other enzyme from the bacterial hormone-sensitive lipase family, probably echoing the specific structural features of the participating subunits. Biophysical studies including analytical gel filtration and ultracentrifugation support the dimeric nature of LpEst1. Structural and mutational analyses of the substrate-binding pocket and active site together with biochemical studies provided insights for understanding the substrate profile of LpEst1 and suggested for the first time the conserved Asp173, which is adjacent to the nucleophile, as a key element in the stabilization of the loop where the oxyanion hole resides.
The hydrolase fold is one of the most versatile structures in the protein realm according to the diversity of sequences adopting such a three-dimensional architecture. In the present study, we clarified the crystal structure of the carboxylesterase Cest-2923 from the lactic acid bacterium Lactobacillus plantarum WCFS1 refined to 2.1 A resolution, determined its main biochemical characteristics and also carried out an analysis of its associative behaviour in solution. We found that the versatility of a canonical alpha/beta hydrolase fold, the basic framework of the crystal structure of Cest-2923, also extends to its oligomeric behaviour in solution. Thus, we discovered that Cest-2923 exhibits a pH-dependent pleomorphic behaviour in solution involving monomers, canonical dimers and tetramers. Although, at neutral pH, the system is mainly shifted to dimeric species, under acidic conditions, tetrameric species predominate. Despite these tetramers resulting from the association of canonical dimers, as is commonly found in many other carboxylesterases from the hormone-sensitive lipase family, they can be defined as 'noncanonical' because they represent a different association mode. We identified this same type of tetramer in the closest relative of Cest-2923 that has been structurally characterized: the sugar hydrolase YeeB from Lactococcus lactis. The observed associative behaviour is consistent with the different crystallographic results for Cest-2923 from structural genomics consortia. Finally, the presence of sulfate or acetate molecules (depending on the crystal form analysed) in the close vicinity of the nucleophile Ser116 allows us to identify interactions with the putative oxyanion hole and deduce the existence of hydrolytic activity within Cest-2923 crystals. STRUCTURED DIGITAL ABSTRACT: Cest-2923 and Cest-2923 bind by x-ray crystallography (1, 2) Cest-2923 and Cest-2923 bind by cosedimentation in solution (1, 2) DATABASE: The atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers: 4BZW for Cest-2923 from native crystals not soaked with substrates (P63 22 space group); 4C01 for Cest-2923 from crystals soaked with phenyl acetate (C2 space group); 4BZZ for Cest-2923 from crystals soaked with isopropenyl acetate (P622 space group).
Q88Y25_Lacpl is an esterase produced by the lactic acid bacterium Lactobacillus plantarum WCFS1 that shows amino-acid sequence similarity to carboxylesterases from the hormone-sensitive lipase family, in particular the AFEST esterase from the archaeon Archaeoglobus fulgidus and the hyperthermophilic esterase EstEI isolated from a metagenomic library. N-terminally His(6)-tagged Q88Y25_Lacpl has been overexpressed in Escherichia coli BL21 (DE3) cells, purified and crystallized at 291 K using the hanging-drop vapour-diffusion method. Mass spectrometry was used to determine the purity and homogeneity of the enzyme. Crystals of His(6)-tagged Q88Y25_Lacpl were prepared in a solution containing 2.8 M sodium acetate trihydrate pH 7.0. X-ray diffraction data were collected to 2.24 A resolution on beamline ID29 at the ESRF. The apparent crystal point group was 422; however, initial global analysis of the intensity statistics (data processed with high symmetry in space group I422) and subsequent tests on data processed with low symmetry (space group I4) showed that the crystals were almost perfectly merohedrally twinned. Most probably, the true space group is I4, with unit-cell parameters a = 169.05, b = 169.05, c = 183.62 A.