Underlying Role of Hydrophobic Environments in Tuning Metal Elements for Efficient Enzyme Catalysis

The catalytic functions of metalloenzymes are often strongly correlated with metal elements in the active sites. However, dioxygen-activating nonheme quercetin dioxygenases (QueD) are found with various first-row transition-metal ions when metal swapping inactivates their innate catalytic activity. To unveil the molecular basis of this seemingly promiscuous yet metal specific enzyme, we transformed manganese-dependent QueD into a nickel-dependent enzyme by sequence-and structure-based directed evolution. Although the net effect of acquired mutations was primarily to rearrange hydrophobic residues in the active site pocket, biochemical, kinetic, X-ray crystallographic, spectroscopic, and computational studies suggest that these modifications in the secondary coordination spheres can adjust the electronic structure of the enzyme-substrate complex to counteract the effects induced by the metal substitution. These results explicitly demonstrate that such noncovalent interactions encrypt metal specificity in a finely modulated manner, revealing the underestimated chemical power of the hydrophobic sequence network in enzyme catalysis.

Automated summary

The catalytic activity of metalloenzymes is strongly related to the metal elements in the active sites. Nonheme quercetin dioxygenases (QueD) are known to have different first-row transition-metal ions binding in their active sites, but the enzyme becomes inactive when the metal is substituted. In this study, manganese-dependent QueD was converted into a nickel-dependent enzyme through directed evolution. The acquired mutations primarily rearrange hydrophobic residues in the active site pocket, adjusting the electronic structure of the enzyme-substrate complex to compensate for the effects of metal substitution.