Engineering Substrate Promiscuity of Nucleoside Phosphorylase Via an Insertions-Deletions Strategy

Area of Science:

• Enzymology and biocatalysis
• Metabolic engineering
• Synthetic biology

Background:

• Nucleoside phosphorylases (NPs) are crucial enzymes in nucleoside metabolism.
• They are vital for synthesizing nucleoside analogs difficult to produce by conventional methods.
• NPs are classified into purine nucleoside phosphorylase (PNP) and pyrimidine/uridine nucleoside phosphorylase (PyNP/UP) based on substrate preference.

Purpose of the Study:

• To engineer the substrate promiscuity of a trimeric PNP from *Aneurinibacillus migulanus* (*Am*PNP).
• To broaden the substrate spectrum of *Am*PNP for synthesizing unnatural nucleoside analogs.
• To demonstrate the utility of an insertions-deletions (InDels) strategy for enzyme engineering.

Main Methods:

• Evolutionary information of the NP-I family was utilized.
• An insertions-deletions (InDels) strategy was employed to engineer *Am*PNP.
• The engineered enzyme, *Am*PNPΔS2_V102K, was characterized for its enzymatic activities.

Main Results:

• The engineered *Am*PNPΔS2_V102K exhibited both PNP and UP activities.
• This enzyme demonstrated phosphorylation activity towards arabinose nucleoside, fluorosyl nucleoside, and dideoxyribose.
• Six purine nucleoside analogs were successfully synthesized using the engineered enzyme, replacing a two-enzyme approach.

Conclusions:

• The InDels strategy effectively enhances enzyme substrate promiscuity.
• The engineered *Am*PNPΔS2_V102K broadens the substrate spectrum for nucleoside analog synthesis.
• This study offers insights into PNP catalytic mechanisms and enzyme engineering benefits.