Second-Shell Amino Acid R266 Helps Determine N-Succinylamino Acid Racemase Reaction Specificity in Promiscuous...

Area of Science:

• Enzyme catalysis
• Protein evolution
• Biochemistry

Background:

• Catalytic promiscuity enables enzymes to catalyze non-native reactions, playing a crucial role in the evolution of new enzyme functions.
• Studying this phenomenon can reveal structural elements that predispose enzymes to evolve novel catalytic activities.

Purpose of the Study:

• To identify preadaptive residues in promiscuous enzymes that facilitate the evolution of new functions.
• To investigate the role of a specific residue (R266) in the catalytic mechanism and substrate specificity of a promiscuous N-succinylamino acid racemase/o-succinylbenzoate synthase (NSAR/OSBS) enzyme.

Main Methods:

• Site-directed mutagenesis was used to alter the R266 residue in the Amycolatopsis NSAR/OSBS enzyme.
• Enzyme kinetics and reaction mechanism analysis were performed to assess the impact of the mutation on both NSAR and OSBS activities.

Main Results:

• The R266 residue, conserved in the NSAR/OSBS subfamily, is located near the catalytic residue K263 but does not directly contact the substrate.
• Mutation of R266 to glutamine significantly reduced NSAR activity (1000-fold decrease in proton exchange rate) but only moderately affected OSBS activity.
• The differential impact of the R266 mutation on NSAR and OSBS activities is attributed to distinct catalytic mechanisms involving K263 for each reaction.

Conclusions:

• R266 is critical for the NSAR activity of the enzyme, likely by influencing the proton exchange mechanism involving K263.
• The structural and mechanistic properties associated with R266 represent a preadaptation that likely facilitated the evolution of NSAR activity within the OSBS enzyme family.