Engineered and Native Coenzyme B12-dependent Isovaleryl-CoA/Pivalyl-CoA Mutase

Area of Science:

• Biochemistry
• Enzymology
• Protein Engineering
• Synthetic Biology

Background:

• Adenosylcobalamin-dependent isomerases are crucial for carbon skeleton rearrangements via radical chemistry.
• A previously studied isobutyryl-CoA mutase variant, IcmF, showed low efficiency and instability in catalyzing the isovaleryl-CoA/pivalyl-CoA mutase (PCM) reaction.

Purpose of the Study:

• To engineer a more efficient and stable pivaloyl-CoA mutase (PCM) by modifying the IcmF enzyme.
• To discover and characterize a novel, authentic PCM enzyme for biotechnological applications.

Main Methods:

• Site-directed mutagenesis of IcmF targeting key active site residues.
• Bioinformatic analysis to identify putative PCM enzymes in genomic databases.
• Cloning, expression, and characterization of the identified PCM and its fused variant (PCM-F).

Main Results:

• The IcmF F598A mutant showed a significant increase in PCM activity (∼17-fold) and specificity (∼4000-fold) but was more susceptible to inactivation.
• A novel, authentic AdoCbl-dependent PCM was identified, cloned, and expressed, exhibiting robust PCM activity and resistance to inactivation.
• A fused variant, PCM-F, retained high PCM activity and stability, lacking detectable isobutyryl-CoA mutase activity.

Conclusions:

• The newly discovered PCM enzyme represents a novel 5'-deoxyadenosylcobalamin-dependent acyl-CoA mutase with distinct substrate specificity.
• PCM and its derivative PCM-F demonstrate significant potential for applications in bioremediation, stereospecific synthesis, and the production of valuable chemicals.