Conformational dynamics of Tetracenomycin aromatase/cyclase regulate polyketide binding and enzyme aggregation propensity

The N-terminal domain of Tetracenomycin aromatase/cyclase (TcmN), an enzyme derived from Streptomyces glaucescens, is 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 various pharmaceutical applications. Examples include antibiotics, such as tetracycline, and anticancer drugs, such as doxorubicin. Because TcmN is a promising enzyme for in vitro production of polyketides, it is important to identify conditions that enhance its thermal resistance and optimize its function. TcmN unfolding, stability, and dynamics were evaluated by fluorescence spectroscopy, circular dichroism, nuclear magnetic resonance 15N relaxation experiments, and microsecond molecular dynamics (MD) simulations. TcmN thermal resistance was enhanced at low protein and high salt concentrations, was pH-dependent, and denaturation was irreversible. Conformational dynamics on the μs-ms timescale were detected for residues in the substrate-binding cavity, and two predominant conformers representing opened and closed cavity states were observed in the MD simulations. Based on the results, a mechanism was proposed in which the thermodynamics and kinetics of the TcmN conformational equilibrium modulate enzyme function by favoring ligand binding and avoiding aggregation. Understanding the principles underlying TcmN stability and dynamics may help in designing mutants with optimal properties for biotechnological applications. [Display omitted] •TcmN is a promising enzyme for polyketide production in the biotech industry.•Conditions that enhance the thermal stability of TcmN were identified.•TcmN fluctuates between an open and closed conformation on the μs timescale.•The catalytic cavity of TcmN prefers a less aggregation-prone closed state.