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 Ser...

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Bibliographic Details
Published in:Biochemistry (Moscow) 2020-06, Vol.85 (6), p.709-716
Main Authors: Kryukova, M. V., Petrovskaya, L. E., Novototskaya-Vlasova, K. A., Kryukova, E. A., Yakimov, S. A., Nikolaeva, A. Y., Boyko, K. M., Dolgikh, D. A., Kirpichnikov, M. P.
Format: Article
Language:English
Subjects:
Amino acids
Biochemistry
Biomedical and Life Sciences
Biomedicine
Bioorganic Chemistry
Catalytic activity
Catalytic Domain
Cysteine
Cysteine - chemistry
Cysteine - genetics
Cysteine - metabolism
Cystine
Deoxyribonucleic acid
DNA
Enzyme Assays - methods
Enzymes
Esterase
Esterases - chemistry
Esterases - genetics
Esterases - metabolism
Kinetics
Life Sciences
Lipase
Low temperature
Metagenomics
Microbiology
Models, Molecular
Mutagenesis, Site-Directed - methods
Mutants
Mutation
Permafrost
Permafrost - chemistry
Properties (attributes)
Protection and preservation
Residues
Serine
Sterol Esterase - chemistry
Sterol Esterase - genetics
Sterol Esterase - metabolism
Substrate Specificity
Thermal stability
Thiols
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Summary: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 k cat 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.
ISSN:0006-2979
1608-3040
DOI:10.1134/S0006297920060085