Hormone-sensitive lipases (HSLs) are widely distributed in microorganisms, plants, and animals. Microbial HSLs are classified into two subfamilies, an unnamed new subfamily and the GDSAG motif subfamily. Due to the lack of structural information, the detailed catalytic mechanism of the new subfamily is not yet clarified. Based on sequence analysis, we propose to name the new subfamily as the GTSAG motif subfamily. We identified a novel HSL esterase E25, a member of the GTSAG motif subfamily, by functional metagenomic screening, and resolved its structure at 2.05 A. E25 is mesophilic (optimum temperature at 50 degrees C), salt-tolerant, slightly alkaline (optimum pH at 8.5) for its activity, and capable of hydrolyzing short chain monoesters (C2-C10). E25 tends to form dimers both in the crystal and in solution. An E25 monomer contains an N-terminal CAP domain, and a classical alpha/beta hydrolase-fold domain. Residues Ser186, Asp282, and His312 comprise the catalytic triad. Structural and mutational analyses indicated that E25 adopts a dimerization pattern distinct from other HSLs. E25 dimer is mainly stabilized by an N-terminal loop intersection from the CAP domains and hydrogen bonds and salt bridges involving seven highly conserved hydrophilic residues from the catalytic domains. Further analysis indicated that E25 also has some catalytic profiles different from other HSLs. Dimerization is essential for E25 to exert its catalytic activity by keeping the accurate orientation of the catalytic Asp282 within the catalytic triad. Our results reveal the structural basis for dimerization and catalysis of an esterase from the GTSAG motif subfamily of the HSL family.
        
Title: Identification of a new subfamily of salt-tolerant esterases from a metagenomic library of tidal flat sediment Jeon JH, Lee HS, Kim JT, Kim SJ, Choi SH, Kang SG, Lee JH Ref: Applied Microbiology & Biotechnology, 93:623, 2012 : PubMed
To search for novel lipolytic enzymes, a metagenomic library was constructed from the tidal flat sediment of Ganghwa Island in South Korea. By functional screening using tributyrin agar plates, 3 clones were selected from among the 80,050 clones of the fosmid library. The sequence analysis revealed that those clones contained different open reading frames, which showed 50-57% amino acid identity with putative lipolytic enzymes in the database. Based on the phylogenetic analysis, they were identified to encode novel members, which form a distinct and new subfamily in the family IV of bacterial lipolytic enzymes. The consensus sequence, GT(S)SA(G)G, encompassing the active site serine of the enzymes was different from the GDSAG motif, conserved in the other subfamily. The genes were expressed in Escherichia coli and recombinant proteins were purified as active soluble forms. The enzymes showed the highest activity toward p-nitrophenyl valerate (C5) and exhibited optimum activities at mesophilic temperature ranges and slightly alkaline pH. In particular, the enzymes displayed salt tolerance with over 50% of the maximum activity remained in the presence of 3 M NaCl (or KCl). In this study, we demonstrated that the metagenomic approach using marine tidal flat sediment as a DNA source expanded the diversity of lipolytic enzyme-encoding genes.
        
Representative scheme of GTSAGmotif structure and an image from PDBsum server
Databases
PDB-Sum
4Q05 Previously Class, Architecture, Topology and Homologous superfamily - PDB-Sum server
FSSP
4Q05Fold classification based on Structure-Structure alignment of Proteins - FSSP server