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The E. coli L-asparaginase V27T mutant: structural and functional characterization and comparison with theoretical
Pawel Strzelczyk1, Di Zhang1, Alexander Wlodawer1
1Center for Structural Biology, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA.
FEBS Letters
|October 31, 2022
Summary
The V27T mutant of Escherichia coli type II L-asparaginase (EcAII) shows reduced glutaminolytic activity but unchanged asparaginolytic activity and stability compared to the wild-type enzyme. Structural analysis reveals altered substrate binding modes.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Bacterial L-asparaginases are established anticancer drugs.
- A theoretical study proposed a V27T mutant of Escherichia coli type II L-asparaginase (EcAII(V27T)) with potentially improved pharmaceutical properties.
Purpose of the Study:
- To experimentally characterize the biophysical and catalytic properties of EcAII(V27T).
- To validate theoretical postulates regarding EcAII(V27T)'s stability and glutaminolytic activity.
Main Methods:
- Experimental characterization of enzyme activity (asparaginolytic and glutaminolytic).
- Stability assays for wild-type EcAII (EcAII(wt)) and EcAII(V27T).
- Crystal structure determination of enzyme-substrate complexes.
Main Results:
- EcAII(V27T) exhibited a fourfold reduction in glutaminolytic activity compared to EcAII(wt).
- Asparaginolytic activity and enzyme stability remained comparable between EcAII(V27T) and EcAII(wt).
- Crystal structures revealed distinct substrate binding modes for both L-Asp and L-Glu in the mutant.
Conclusions:
- EcAII(V27T) possesses reduced glutaminolytic activity, a desirable trait for anticancer applications.
- The V27T mutation does not significantly enhance enzyme stability but alters substrate binding.
- Experimental findings partially support and refine theoretical predictions for EcAII(V27T).
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