Effect of Cysteine Residue Substitution in the GCSAG Motif of the PMGL2 Esterase Active Site on the Enzyme Properties


The gene coding for PMGL2 esterase, which belongs to the family of mammalian hormone-sensitive lipases (HSLs), was discovered by screening a metagenomic DNA library from a permafrost soil. The active site of PMGL2 contains conserved GXSXG motif which includes Cys173 residue next to the catalytic Ser174. In order to clarify the functional role of the cysteine residue in the GCSAG motif, we constructed a number of PMGL2 mutants with Cys173 substitutions and studied their properties. The specific activity of the C173D mutant exceeded the specific activity of the wild-type enzyme (wtPMGL2) by 60%, while the C173T/C202S mutant displayed reduced catalytic activity. The activity of the C173D mutant with p-nitrophenyl octanoate was 15% higher, while the activity of the C173T/C202S mutant was 17% lower compared to wtPMGL2. The C173D mutant was also characterized by a high activity at low temperatures (20-35°C) and significant loss of thermal stability. The kcat value for this protein was 56% higher than for the wild-type enzyme. The catalytic constants of the C173S mutant were close to those of wtPMGL2; this enzyme also demonstrated the highest thermal stability among the studied mutants. The obtained results demonstrate that substitutions of amino acid residues adjacent to the catalytic serine residue in the GXSXG motif can have a significant effect on the properties of PMGL2 esterase.

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mammalian hormone-sensitive lipase


polyethylene glycol


splicing by overlapping extension PCR


wild-type PMGL2 esterase


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This work was partially supported by the Russian Foundation for Basic Research (project No. 18-04-00491) and by the Molecular and Cellular Biology Program of the Russian Academy of Sciences.

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Correspondence to L. E. Petrovskaya.

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This article does not contain any studies with human participants or animals performed by any of the authors. The authors declare no conflict of interest in financial or any other sphere.

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Kryukova, M., Petrovskaya, L., Novototskaya-Vlasova, K. et al. Effect of Cysteine Residue Substitution in the GCSAG Motif of the PMGL2 Esterase Active Site on the Enzyme Properties. Biochemistry Moscow 85, 709–716 (2020). https://doi.org/10.1134/S0006297920060085

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  • PMGL2 esterase
  • HSL family
  • GCSAG motif
  • mutagenesis
  • thermal stability
  • three-dimensional structure