Characterization of alkaline phosphatase PhoK from Sphingomonas sp. BSAR-1 for phosphate monoester synthesis and hydrolysis

• Published in: Biochimica et biophysica acta. Proteins and proteomics Vol. 1868; no. 1; p. 140291
• Main Authors: Lukesch, Michael, Tasnádi, Gábor, Ditrich, Klaus, Hall, Mélanie, Faber, Kurt
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2020
• Subjects: Alkaline Phosphatase - chemistry; Alkaline Phosphatase - genetics; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Biocatalysis; Biotransformation; Enzyme; Esters - chemistry; Hydrolysis; Phosphatase; Phosphate ester; Phosphates - chemistry; Phosphorylation; Sphingomonas - enzymology; Threonine - chemistry; Transphosphorylation; Transphosphorylation; Phosphate ester; Phosphatase; Enzyme; Biotransformation
• ISSN: 1570-9639; 1878-1454
• DOI: 10.1016/j.bbapap.2019.140291

The biocatalytic activity of a so far underexploited alkaline phosphatase, PhoK from Sphingomonas sp. BSAR-1, was extensively studied in transphosphorylation and hydrolysis reactions. The use of high-energy phosphate donors and oligophosphates as suitable phosphate donors was evaluated, as well as the hydrolytic activity on a variety of phosphate monoesters. While substrates bearing free hydroxy group displayed only moderate reactivity as acceptors for transphosphorylation by PhoK, strong hydrolytic activity on a broad variety of phosphate monoesters under alkaline conditions was observed. Site-directed mutagenesis of selected amino acid residues in the active site provided valuable insights on their involvement in enzyme catalysis. The key residue Thr89 so far postulated to engage in enzyme phosphorylation was confirmed to be crucial for catalysis and could be replaced by serine, albeit with much lower catalytic efficiency. •Biocatalytic potential of PhoK was evaluated for synthetic applications.•Phosphotransfer was found on a variety of substrates using pyrophosphate as donor.•Strong hydrolytic activity on a range of phosphate monoesters was observed.•Key residue Thr89 was confirmed to be crucial for catalysis.