(117a) Elucidating the Complex Interactions between Stability, Oligomericity, and Activity of ?-Amino Ester Hydrolase

α-Amino ester hydrolases (AEHs, EC 3.1.1.43) comprise a small class of enzymes capable of enantioselective production of semi-synthetic β-lactam antibiotics. AEH activity was first described in 1972 by Takahashi et al. [1], but AEHs from A. turbidans [2], X. citri [3], and X. campestris pv. campestris [4], have only recently been isolated, expressed in E. coli, and characterized in terms of substrate specificity, solvent effects, and mutability. Despite their rapid kinetics and high selectivity, AEH use is complicated by rapid deactivation and low stability [5]. Improvements in AEH stability has so far been limited, and further insight into the nature of AEH in solution is necessary.

Several studies have shown that X. citri and A. turbidans AEH monomers associate in a tetrameric structure in crystalline form. X. citri AEH monomers have also been shown to associate as a tetramer in solution, and dissociation of the tetramer into AEH monomers eliminates activity [6]. The tetrameric association has been assumed to be present and necessary for activity in all AEHs; however analytical ultracentrifugation (AUC) studies of X. campestris AEH show that only 15% of AEH in solution appears as a tetramer under conditions where the enzyme is also active. Furthermore, mutant AEHs designed for increased monomer stability demonstrate higher tetrameric association (up to 30%) as well as up to 70% higher activity toward cephalexin hydrolysis under the same conditions. This suggests that unlike previous studies, the AEH monomer is active, but association exists between monomer stability, tetrameric association, and kinetic activity.

This study provides further insight into the relationship between the stability, oligomericity, and activity of X. campestris AEH by comparing the deactivation kinetics of the wildtype AEH to mutant AEHs of varying stability and oligomericity. In addition, AEH tetramer was isolated from AEH monomer, and the activity of both forms were analyzed. Several mutants were analyzed using differential scanning fluorimetry (DSF) to determine melting temperatures of the monomers (T_m), ampicillin hydrolysis assays to determine activity levels and deactivation kinetics, and AUC to determine oligomeric states. Finally, AUC was used to determine oligomeric fractions of A. turbidans and X. citri AEHs in solution to compare along with literature data of kinetics and stability to X. campestris AEH. The relationships between stability, oligomericity, and activity as well as comparisons among the class of AEHs is discussed herein.

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