Crystal structure of PMGL2 esterase from the hormone-sensitive lipase family with GCSAG motif around the catalytic serine

Abstract

Lipases comprise a large class of hydrolytic enzymes which catalyze the cleavage of the ester bonds in triacylglycerols and find numerous biotechnological applications. Previously, we have cloned the gene coding for a novel esterase PMGL2 from a Siberian permafrost metagenomic DNA library. We have determined the 3D structure of PMGL2 which belongs to the hormone-sensitive lipase (HSL) family and contains a new variant of the active site motif, GCSAG. Similar to many other HSLs, PMGL2 forms dimers in solution and in the crystal. Our results demonstrated that PMGL2 and structurally characterized members of the GTSAG motif subfamily possess a common dimerization interface that significantly differs from that of members of the GDSAG subfamily of known structure. Moreover, PMGL2 had a unique organization of the active site cavity with significantly different topology compared to the other lipolytic enzymes from the HSL family with known structure including the distinct orientation of the active site entrances within the dimer and about four times larger size of the active site cavity. To study the role of the cysteine residue in GCSAG motif of PMGL2, the catalytic properties and structure of its double C173T/C202S mutant were examined and found to be very similar to the wild type protein. The presence of the bound PEG molecule in the active site of the mutant form allowed for precise mapping of the amino acid residues forming the substrate cavity.

Structural and Biochemical Characterization of a Cold-Active PMGL3 Esterase with Unusual Oligomeric Structure

“…The bacterial HSL family is divided into two subfamilies based on G(D/E)SAG or G(T/X)SAG motifs of the active site, with various amino acids present in the second position of the latter group [ 34 , 35 , 36 ]. As PMGL3 contains 147 GESAG 151 motif, comparison of its structure with those of homologue enzymes revealed a number of structurally similar bHSL enzymes from GDSAG subfamily ( Table 7 ).…”

“…Oligomeric quaternary structure is a characteristic feature of many esterases belonging to the bHSL family [ 35 , 36 , 37 , 38 ] with dimeric state being quite common. It was proposed previously that dimerization interface of bHSLs differs between enzymes of GDSAG and GTSAG subfamilies [ 35 , 36 ]. Thus, for GDSAG subfamily the dimeric interface is mainly formed by the interactions between two antiparallel β8-strands from each of the subunits and doesn’t involve the CAP-domain [ 39 , 40 , 41 ].…”

“…A major difference between GDSAG and GT/XSAG bHSL subfamilies is the mutual orientation of β8 strands within the dimeric interface. They are antiparallel in case of GDSAG subfamily [ 39 , 40 , 41 ] and about to perpendicular for GT/XSAG [ 35 , 36 ]. According to our results, we speculate that bHSLs of GDSAG subfamily with dimeric and/or tetrameric organization could be further subdivided based on details of their dimeric interface.…”

“…The active site residues of PMGL3 also possess a canonical conformation. Oligomeric quaternary structure is a characteristic feature of many esterases belonging to the bHSL family [35][36][37][38] with dimeric state being quite common. It was proposed previously that dimerization interface of bHSLs differs between enzymes of GDSAG and GTSAG subfamilies [35,36].…”

“…Oligomeric quaternary structure is a characteristic feature of many esterases belonging to the bHSL family [35][36][37][38] with dimeric state being quite common. It was proposed previously that dimerization interface of bHSLs differs between enzymes of GDSAG and GTSAG subfamilies [35,36]. Thus, for GDSAG subfamily the dimeric interface is mainly Oligomeric quaternary structure is a characteristic feature of many esterases belonging to the bHSL family [35][36][37][38] with dimeric state being quite common.…”