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Identification and structure‐guided enzyme engineering of an extremophilic protein phosphatase
Author(s) -
Chen Tiantian,
Vander Kooi Carl D.,
Anwar Humza,
Sharma Savita,
Murphy Robert D.,
Gentry Matthew S.,
Vander Kooi Craig W.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.03788
Subject(s) - phosphatase , biochemistry , dual specificity phosphatase , protein tyrosine phosphatase , enzyme , glucan , biology , chemistry
Glucan phosphatases are members of the dual‐specificity phosphatase (DSP) family of cysteine‐dependent phosphatases. In plants, glucan phosphatases regulate starch metabolism while in animals they regulate glycogen metabolism. All known glucan phosphatase family members possess a DSP with a highly conserved active site. There is significant interest in developing glucan phosphatases for utilization in industrial processes involving starch and other polyglucans. We identified a putative phosphatase from the thermophilic bacterium Thermogutta terrifontis (Tt‐phos). Tt‐phos was expressed, purified and found to have a high thermal stability and tyrosine phosphatase activity. Determination of the crystal structure of Tt‐phos demonstrated that it adopts a glucan phosphatase‐like fold and provided key details of its active site architecture. Structure‐guided design was utilized to engineer the enzyme to produce a novel chimeric enzyme that has robust glucan phosphatase activity at elevated temperature. This study establishes the structural mechanism of Tt‐phos and provides key insights into the relationship of glucan phosphatase to the broader phosphatase family. Further, these data demonstrate a successful application of protein engineering for industrial applications of glucan phosphatases. Support or Funding Information This work was funded by the NSF (CHE CLP:1808304). Data was collected at the Advanced Photon Source (APS) SER‐CAT.