(117a) Elucidating the Complex Interactions between Stability, Oligomericity, and Activity of ?-Amino Ester Hydrolase
- Conference: AIChE Annual Meeting
- Year: 2022
- Proceeding: 2022 Annual Meeting
- Group: Food, Pharmaceutical & Bioengineering Division
- Time: Monday, November 14, 2022 - 12:30pm-12:48pm
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 . 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 (Tm), 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.
- Takahashi, T., et al., Enzymatic synthesis of cephalosporins. J Am Chem Soc, 1972. 94(11): p. 4035-7.
- Polderman-Tijmes, J.J., et al., Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding an alpha-amino acid ester hydrolase from Acetobacter turbidans. Appl Environ Microbiol, 2002. 68(1): p. 211-8.
- Barends, T.R.M., et al., The Sequence and Crystal Structure of the Î±-Amino Acid Ester Hydrolase fromXanthomonas citriDefine a New Family of Î²-Lactam Antibiotic Acylases. Journal of Biological Chemistry, 2003. 278(25): p. 23076-23084.
- Blum, J.K. and A.S. Bommarius, Amino ester hydrolase from Xanthomonas campestris pv. campestris, ATCC 33913 for enzymatic synthesis of ampicillin. J Mol Catal B Enzym, 2010. 67(1-2): p. 21-28.
- Blum, J.K., M.D. Ricketts, and A.S. Bommarius, Improved thermostability of AEH by combining B-FIT analysis and structure-guided consensus method. J Biotechnol, 2012. 160(3-4): p. 214-21.
- Kato, K. and A. Kakinuma, Dissociation and Reassociation of Xanthomonas Alpha-Amino-Acid Ester Hydrolase. Agricultural and Biological Chemistry, 1980. 44(7): p. 1663-1664.