Altered Cofactor Preference of Thermostable StDAPDH by a Single Mutation at K159

Area of Science:

• Biocatalysis and Enzyme Engineering
• Synthetic Biology
• Protein Chemistry

Background:

• D-amino acid production via reductive amination of 2-keto acids offers high yields and environmental benefits.
• meso-diaminopimelate dehydrogenase (meso-DAPDH) from Symbiobacterium thermophilum (StDAPDH) is a thermostable enzyme catalyzing this reaction but uses NADP(H), which is less industrially prevalent than NAD(H).
• Cofactor engineering of StDAPDH is necessary to broaden its industrial applicability.

Purpose of the Study:

• To engineer StDAPDH for cofactor specificity using NAD(H) instead of NADP(H).
• To investigate the role of specific amino acid residues in cofactor binding and specificity.
• To provide insights into the catalytic mechanism and guide future enzyme modifications.

Main Methods:

• Site-directed mutagenesis targeting conserved lysine residues in the cofactor binding site of StDAPDH.
• Enzyme activity assays using various keto acids and cofactors (NADH and NADPH).
• Molecular dynamics simulations to analyze the structural and dynamic effects of mutations on cofactor interaction.

Main Results:

• Mutation of lysine residues, specifically K159R, altered cofactor specificity, enabling activity with NADH.
• The K159R mutant exhibited equal affinity for NADH and NADPH with pyruvic acid as the substrate.
• Molecular dynamics simulations indicated that arginine's steric bulk and salt bridge interactions with NADH stabilize the active conformation of the K159R mutant.

Conclusions:

• Lysine residues in the cofactor binding loop are critical for StDAPDH's native cofactor specificity.
• The K159R mutation successfully engineered StDAPDH to utilize the more common NADH cofactor.
• Structural insights from simulations explain the enhanced activity and stability with NADH, guiding further enzyme engineering efforts for industrial biocatalysis.