Biophysical characterization of the N-terminal domain of Tetracenomycin ARO/CYC

• Autor(a) principal: Veronica Silva Valadares
• Data de Publicação: 2020
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Institucional da UFMG
• Texto Completo: http://hdl.handle.net/1843/53996; https://orcid.org/0000-0003-1931-3464
• topic: Tetracenomycin; TcmN; aromatase; cyclase; ARO/CYC; NMR; polyketides; cyclization; aromatization; Bioquímica; Aromatase; Ciclização; Policetídeos; Tetraciclina

Tetracenomycin aromatase/cyclase (Tcm) is an enzyme derived from Streptomyces glaucescens involved in polyketide cyclization, aromatization, and folding. Polyketides are a diverse class of secondary metabolites produced by certain groups of bacteria, fungi, and plants with pharmaceutical applications. Examples include antibiotics, such as tetracycline, and anticancer drugs, such as doxorubicin. The N-terminal domain of Tcm (TcmN) participates in the specific cyclization of polyketides, and is classified as belonging to the Bet v 1-like superfamily. TcmN catalyzes the formation of the first (C9-C142) and second (C7-C16) rings of polyketides in vivo and in vitro. This work characterized the stability and conformational diversity TcmN, which is important for its possible use in industrial applications, such as engineering polyketide biosynthesis. TcmN was expressed in Escherichia coli BL21(DE3) and purified. Secondary structure and stability were assessed in different buffer conditions by circular dichroism (CD) and fluorescence spectroscopy. Nuclear Magnetic Resonance (NMR) spectra were acquired at different field strengths (900, 800, and 600 MHz). 6 x 1 μs Molecular Dynamics (MD) simulations of TcmN were performed in explicit solvent. Thermal denaturation of TcmN is irreversible, and the denaturation temperature is reduced at higher concentrations. TcmN is most stable around pH 8, and in the presence of NaI and NaCl. NMR and MD findings suggest that substrate binding and product release can be modulated by flexibility of specific loop segments. Together, the results indicate that TcmN exists in equilibrium between an open conformation that favors ligand binding in the main hydrophobic cavity, and a closed state that protects it against solvent exposure and aggregation.