Flavin-dependent hydroxylases (FdHs) play an essential role in various biosynthetic pathways, hydroxylating phenols with impeccable site- and stereoselectivity. Thus, FdH biocatalysts have immense potential to facilitate synthesis of drug-like molecules. A detailed understanding of key catalytic steps is essential for rational manipulation of FdH function to improve biocatalytic utility. In class A hydroxylases, one such step – flavin reduction – occurs efficiently only after binding an appropriate substrate. While this function is characteristic of class A FdHs, the mechanism underlying the enhancement of flavin reduction is poorly understood. Crystal structures of potential biocatalyst TropB, a class A FdH, show the “flavin-in” position when unbound, but density for both “in” and “out” configurations in the substrate-bound structure, suggesting that substrate binding may enhance flavin reduction by altering flavin dynamics. This work investigates enzyme and substrate characteristics that are important for flavin motion and reduction in TropB.<br/><br/>A converged, minimum free energy path for flavin conformational change in TropB with native substrate (3-methyl-orcinaldehyde) bound was generated using the harmonic Fourier bead string method. This approach enabled the description of multiple changing degrees of freedom through one collective variable defined broadly to include 463 atoms. The final path was characterized by molecular dynamics simulations, which were used to investigate the free energy surface along the transition. Further analysis illustrates the roles of the substrate and key residues Arg206 and Trp299 in flavin movement and provides a thermodynamic rationale for flavin motion in the presence of substrate. These models can additionally be used to examine the NADPH binding interface and the impact of other factors on flavin motion, including flavin oxidation state, substrate charge, and mutation of Arg206. Evaluation of flavin properties, substrate characteristics, and protein residues that affect flavin conformational change will improve insight into this enzyme class and guide rational redesign of FdHs.

Exploring flavin dynamics in biocatalyst tropB with theory and molecular modeling

ACS Fall 2021

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