Evolution of Conformational Dynamics Determines the Conversion of a Promiscuous Generalist into a Specialist Enzyme Zou, Taisong Risso, Valeria Alejandra Gavira Gallardo, José Antonio Sánchez Ruiz, José Manuel Ozkan, S. Banu Protein dynamics and structure Ancestral enzyme Molecular dynamics Center for Biological Physics, Department of Physics, Arizona State University 2Departamento de Qu ımica F ısica, Facultad de Ciencias, Universidad de Granada, Granada, Spain 3Laboratorio de Estudios Cristalogr aficos, Instituto Andaluz de Ciencias de la Tierra (Consejo Superior de Investigaciones Cient ıficas—Universidad de Granada), Granada, Spain This work was supported by grants BIO2012-34937, CSD2009- 00088, BIO2010-16800, 1U54GN0945999, and “Factor ıa Espa~nola de Cristalizaci on,” Consolider-Ingenio 2010 from Spanish Ministry of Economy and Competitiveness, FEDER Funds. The authors thank the ESRF staff in Grenoble (France) for excellent support during collection of the data required to obtain the structures shown in figure 1. S.B.O. and T.Z. acknowledge XSEDE computing time and CLAS/ASU award. β-lactamases are produced by many modern bacteria as a mechanism of resistance toward β-lactam antibiotics, the most common antibiotics in use. β-lactamases, however, are ancient enzymes that originated billions of years ago. Recently, proteins corresponding to 2- to 3-Gy-old Precambrian nodes in the evolution of Class A β-lactamases have been prepared and shown to be moderately efficient promiscuous catalysts, able to degrade a variety of antibiotics with catalytic efficiency levels similar to those of an average modern enzyme. Remarkably, there are few structural differences (in particular at the active-site regions) between the resurrected enzymes and a penicillin-specialist modern β-lactamase. Here, we propose that the ancestral promiscuity originates from conformational dynamics. We investigate the differences in conformational dynamics of the ancient and extant β-lactamases through MD simulations and quantify the contribution of each position to functionally related dynamics through Dynamic Flexibility Index. The modern TEM-1 lactamase shows a comparatively rigid active-site region, likely reflecting adaptation for efficient degradation of a specific substrate (penicillin), whereas enhanced deformability at the active-site neighborhood in the ancestral resurrected proteins likely accounts for the binding and subsequent degradation of antibiotic molecules of different size and shape. Clustering of the conformational dynamics on the basis of Principal Component Analysis is in agreement with the functional divergence, as the ancient β-lactamases cluster together, separated from their modern descendant. Finally, our analysis leads to testable predictions, as sites of potential relevance for the evolution of dynamics are identified and mutations at those sites are expected to alter substrate-specificity. 2024-01-18T12:52:03Z 2024-01-18T12:52:03Z 2015-01 info:eu-repo/semantics/article Taisong Zou, Valeria A. Risso, Jose A. Gavira, Jose M. Sanchez-Ruiz, S. Banu Ozkan, Evolution of Conformational Dynamics Determines the Conversion of a Promiscuous Generalist into a Specialist Enzyme, Molecular Biology and Evolution, Volume 32, Issue 1, January 2015, Pages 132–143, https://doi.org/10.1093/molbev/msu281 https://hdl.handle.net/10481/86924 10.1093/molbev/msu281 eng http://creativecommons.org/licenses/by-nc-nd/4.0/ info:eu-repo/semantics/openAccess Attribution-NonCommercial-NoDerivatives 4.0 Internacional OXFORD UNIV PRESS