Characterization Of Thermodynamics, Kinetics, And Binding Mechanism Of The Interaction Between Tetracenomycin Aromatase/Cyclase (TcmN) And Naringenin By NMR Titration Experiments

Introduction: The N-terminal domain of Tetracenomycin aromatase/cyclase (TcmN) is an enzyme from Streptomyces glaucescens directly involved in the cyclization, aromatization, and folding of polyketides. Aromatic polyketides are an important class of secondary metabolites with a wide range of biological activities. Because TcmN is a promising enzyme for in vitro production of polyketides, understanding the interaction of TcmN with substrates, intermediates, and products is vital for comprehending the enzyme's function. Objectives: A proposed mechanism suggests that the thermodynamics and conformational equilibrium of TcmN play a role in modulating its enzymatic function, promoting ligand binding, and facilitating product release (BBA, v. 1865, n. 9, p. 129949, 2021). Our goal is to comprehensively characterize the TcmN interaction with naringenin. Through structural, thermodynamic, and kinetic analyses, we aim to elucidate the binding mechanism between TcmN and naringenin, an analog of the two-ring intermediate in the TcmN-catalyzed reaction. Material and Methods: 1H-15N HSQC and 1H-15N sofast-HMQC NMR experiments of 15N-labeled TcmN titrated with different naringenin concentrations were acquired. Structural changes in TcmN upon naringenin titration were assessed through chemical shift perturbation (CSP) analysis. To gain further insights into the thermodynamics, kinetics, and molecular recognition mechanism of the interaction, NMR lineshape analysis was performed using the Matlab-based software Titan (Sci. Rep., v. 6, n. 1, p. 24826, 2016). Results: Comparison between simulated NMR spectra and experimental ones, with a focus on 1H and 15N NMR chemical shifts and lineshapes, allowed for the evaluation of different binding mechanisms. CSP analysis indicated that residues from the catalytic cavity were more affected by the interaction with naringenin. Residues distant from the catalytic site were also perturbed suggesting structural allostery triggered by the interaction. Lineshape analysis of the titration spectra indicated that a simple two-state binding mechanism was inadequate to describe the TcmN-ligand interaction. Instead, data fitting and spectra simulation revealed that a sequential binding mechanism provided a more accurate description of the interaction. The structural position of the residues with higher CSP suggests that the first naringenin is positioned further inside the cavity, and the second is close to the entrance of the pocket, interacting with conserved residues that may govern putative substrate-binding and product-exit modes.

This work was supported by CNPq, FAPEMIG, and CAPES. Further acknowledgements: Laboratório de Ressonância Magnética Nuclear de Alta Resolução da UFMG – LAREMAR; HMS Bio-molecular NMR Facility and DFCI NMR Core