Expression and kinetic characterization of PYCR3

Area of Science:

• Biochemistry
• Enzymology
• Cancer Metabolism

Background:

• Proline biosynthesis enzymes (PYCRs) are crucial in human metabolism.
• PYCR1 is upregulated in many cancers, impacting cancer cell metabolism.
• PYCR3 is the least studied isoform due to expression challenges.

Purpose of the Study:

• To establish a robust recombinant expression system for human PYCR3.
• To characterize the enzymatic activity and kinetics of PYCR3.
• To evaluate the isoform specificity of potential PYCR inhibitors.

Main Methods:

• Expression of soluble SUMO-PYCR3 in Escherichia coli.
• Purification of the SUMO-PYCR3 fusion protein and subsequent tag removal.
• Bi-substrate kinetic measurements and product inhibition studies.
• Screening of proline analogs for PYCR3 inhibition.

Main Results:

• A functional expression system for soluble PYCR3 was established.
• PYCR3 exhibits activity with both NADPH and NADH, following a random ordered bi bi mechanism.
• N-formyl-l-proline, a PYCR1 inhibitor, shows 10-fold greater selectivity for PYCR1 over PYCR3.

Conclusions:

• The developed SUMO-PYCR3 expression system facilitates further research on this enzyme.
• Understanding PYCR3's role in cancer metabolism can reveal new therapeutic targets.
• The isoform-specific inhibition data are critical for designing targeted cancer therapies.