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1. Pervasive Pairwise Intragenic Epistasis among Sequential Mutations in TEM-1 β-Lactamase

   https://doi.org/10.1016/j.jmb.2019.03.020  

   Gonzalez, Courtney E.; Ostermeier, Marc (May 2019, Journal of Molecular Biology)
2. Machine Learning Classification Model for Functional Binding Modes of TEM-1 β-Lactamase

   https://doi.org/10.3389/fmolb.2019.00047  

   Wang, Feng; Shen, Li; Zhou, Hongyu; Wang, Shouyi; Wang, Xinlei; Tao, Peng (July 2019, Frontiers in Molecular Biosciences)
3. The Role of Rigid Residues in Modulating TEM-1 β-Lactamase Function and Thermostability

   https://doi.org/10.3390/ijms22062895  

   Kolbaba-Kartchner, Bethany; Kazan, I. Can; Mills, Jeremy H.; Ozkan, S. Banu (March 2021, International Journal of Molecular Sciences)

   null (Ed.)

   The relationship between protein motions (i.e., dynamics) and enzymatic function has begun to be explored in β-lactamases as a way to advance our understanding of these proteins. In a recent study, we analyzed the dynamic profiles of TEM-1 (a ubiquitous class A β-lactamase) and several ancestrally reconstructed homologues. A chief finding of this work was that rigid residues that were allosterically coupled to the active site appeared to have profound effects on enzyme function, even when separated from the active site by many angstroms. In the present work, our aim was to further explore the implications of protein dynamics on β-lactamase function by altering the dynamic profile of TEM-1 using computational protein design methods. The Rosetta software suite was used to mutate amino acids surrounding either rigid residues that are highly coupled to the active site or to flexible residues with no apparent communication with the active site. Experimental characterization of ten designed proteins indicated that alteration of residues surrounding rigid, highly coupled residues, substantially affected both enzymatic activity and stability; in contrast, native-like activities and stabilities were maintained when flexible, uncoupled residues, were targeted. Our results provide additional insight into the structure-function relationship present in the TEM family of β-lactamases. Furthermore, the integration of computational protein design methods with analyses of protein dynamics represents a general approach that could be used to extend our understanding of the relationship between dynamics and function in other enzyme classes. 

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4. Fitness Effects of Single Amino Acid Insertions and Deletions in TEM-1 β-Lactamase

   https://doi.org/10.1016/j.jmb.2019.04.030  

   Gonzalez, Courtney E.; Roberts, Paul; Ostermeier, Marc (May 2019, Journal of Molecular Biology)
5. Time-resolved β-lactam cleavage by L1 metallo-β-lactamase

   https://doi.org/10.1038/s41467-022-35029-3  

   Wilamowski, M.; Sherrell, D. A.; Kim, Y.; Lavens, A.; Henning, R. W.; Lazarski, K.; Shigemoto, A.; Endres, M.; Maltseva, N.; Babnigg, G.; et al (December 2022, Nature Communications)

   Abstract Serial x-ray crystallography can uncover binding events, and subsequent chemical conversions occurring during enzymatic reaction. Here, we reveal the structure, binding and cleavage of moxalactam antibiotic bound to L1 metallo-β-lactamase (MBL) from Stenotrophomonas maltophilia . Using time-resolved serial synchrotron crystallography, we show the time course of β-lactam hydrolysis and determine ten snapshots (20, 40, 60, 80, 100, 150, 300, 500, 2000 and 4000 ms) at 2.20 Å resolution. The reaction is initiated by laser pulse releasing Zn 2+ ions from a UV-labile photocage. Two metal ions bind to the active site, followed by binding of moxalactam and the intact β-lactam ring is observed for 100 ms after photolysis. Cleavage of β-lactam is detected at 150 ms and the ligand is significantly displaced. The reaction product adjusts its conformation reaching steady state at 2000 ms corresponding to the relaxed state of the enzyme. Only small changes are observed in the positions of Zn 2+ ions and the active site residues. Mechanistic details captured here can be generalized to other MBLs. 

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