Modulating phosphate transfer process for promoting phosphorylation activity of acid phosphatase

Area of Science:

• Biochemistry
• Enzyme Engineering
• Synthetic Biology

Background:

• Klebsiella pneumoniae acid phosphatase is crucial for large-scale nucleotide synthesis.
• Substrate phosphate acceptance limits the efficiency of phosphate transfer in enzymatic reactions.

Purpose of the Study:

• To engineer variants of Klebsiella pneumoniae acid phosphatase with improved efficiency for nucleotide synthesis.
• To overcome limitations in substrate interaction and steric hindrance affecting enzyme activity.

Main Methods:

• Protein engineering to create Klebsiella pneumoniae acid phosphatase variants, including the E104G mutant.
• Crystallography and quantum mechanics/molecular mechanics (QM/MM) to analyze enzyme structure and function.
• Enzyme activity assays to evaluate hydrolysis and phosphorylation.
• Genetic modification to control degradation rates of synthesized nucleotides.

Main Results:

• The E104G variant demonstrated significantly enhanced hydrolysis activity while retaining high phosphorylation activity.
• Structural analyses revealed that the E104G mutation improves substrate binding and lowers the activation energy barrier.
• Engineered variants achieved substantially improved conversion rates in nucleotide synthesis.
• Knocking out degradation enzymes effectively controlled the degradation of inosinic acid and guanylic acid.

Conclusions:

• A structure-based design strategy can effectively enhance enzyme specificity and efficiency.
• Engineered Klebsiella pneumoniae acid phosphatase variants show promise for industrial-scale nucleotide synthesis.
• Optimizing enzyme-substrate interactions is key to improving enzymatic conversion rates.