Investigating operational stability and causes of cofactor release in fold type I amine transaminase.

Amine transaminases (ATAs) are pyridoxal phosphate (PLP) dependent biocatalysts frequently employed in chiral amine synthesis. Yields of ATA-catalyzed reactions often suffer from low enzyme operational stability due to weak interactions between the ATA and the aminated cofactor pyridoxamine phosphate (PMP), which can leak from the active site during catalytic cycles, destabilizing the ATA structure and causing inactivation. This study compared the operational stability of a single-substituted Chromobacterium violaceum ATA mutant (CvATA V124N) to its wildtype counterpart (CvATA) and Silicibacter pomeroyi ATA (SpATA) under various operating conditions. A kinetic model was developed to compare the PLP-binding and PMP-release rate constants of ATAs, and molecular dynamics (MD) simulations were performed to uncover the effects of the substitution on cofactor-ATA interactions. High amino donor concentration was found to selectively inhibit aminated product formation while promoting PMP release, and an amino donor-inhibition-induced PMP release mechanism via allosteric regulation was hypothesized based on the multimeric nature of ATA structures. MD simulation results predicted that the PMP release is likely initiated by solvent attack, and the reduced operational stability of the V124N mutant may be explained by the increased hydrophilicity introduced by the substitution. A hydrogen bond network near the SpATA active site, formed by a conserved set of hydrophilic amino acids, was identified via sequence analysis as a rare feature that potentially stabilizes the cofactor binding site structure and reduces solvent access to the solvent-free regions, which may explain the high cofactor affinity and operational stability of the ATA.