New Inhibitors of Laccase and Tyrosinase by Examination of Cross-Inhibition between Copper-Containing Enzymes.

Coppers play crucial roles in the maintenance homeostasis in living species. Approximately 20 enzyme families of eukaryotes and prokaryotes are known to utilize copper atoms for catalytic activities. However, small-molecule inhibitors directly targeting catalytic centers are rare, except for those that act against tyrosinase and dopamine-ß-hydroxylase (DBH). This study tested whether known tyrosinase inhibitors can inhibit the copper-containing enzymes, ceruloplasmin, DBH, and laccase. While most small molecules minimally reduced the activities of ceruloplasmin and DBH, aside from known inhibitors, 5 of 28 tested molecules significantly inhibited the function of laccase, with the Ki values in the range of 15 to 48 µM. Enzyme inhibitory kinetics classified the molecules as competitive inhibitors, whereas differential scanning fluorimetry and fluorescence quenching supported direct bindings. To the best of our knowledge, this is the first report on organic small-molecule inhibitors for laccase. Comparison of tyrosinase and DBH inhibitors using cheminformatics predicted that the presence of thione moiety would suffice to inhibit tyrosinase. Enzyme assays confirmed this prediction, leading to the discovery of two new dual tyrosinase and DBH inhibitors.

Hydroxamic Acid as a Potent Metal-Binding Group for Inhibiting Tyrosinase.

Tyrosinase, a metalloenzyme containing a dicopper cofactor, plays a central role in synthesizing melanin from tyrosine. Many studies have aimed to identify small-molecule inhibitors of tyrosinase for pharmaceutical, cosmetic, and agricultural purposes. In this study, we report that hydroxamic acid is a potent metal-binding group for interacting with dicopper atoms, thereby inhibiting tyrosinase. Hydroxamate-containing molecules, including anticancer drugs targeting histone deacetylase, vorinostat and panobinostat, significantly inhibited mushroom tyrosinase, with inhibitory constants in the submicromolar range. Of the tested molecules, benzohydroxamic acid was the most potent. Its inhibitory constant of 7 nM indicates that benzohydroxamic acid is one of the most potent tyrosinase inhibitors. Results from differential scanning fluorimetry revealed that direct binding mediates inhibition. The enzyme kinetics were studied to assess the inhibitory mechanism of the hydroxamate-containing molecules. Experiments with B16F10 cell lysates confirmed that the new inhibitors are inhibitory against mammalian tyrosinase. Docking simulation data revealed intermolecular contacts between hydroxamate-containing molecules and tyrosinase.

Microbial rhodoquinone biosynthesis proceeds via an atypical RquA-catalyzed amino transfer from S-adenosyl-L-methionine to ubiquinone.

Rhodoquinone (RQ) is a close analogue of ubiquinone (UQ) that confers diverse bacterial and eukaryotic taxa the ability to utilize fumarate as an electron acceptor in hypoxic conditions. The RquA protein, identified in a Rhodospirillum rubrum RQ-deficient mutant, has been shown to be required for RQ biosynthesis in bacteria. In this report, we demonstrate that RquA, homologous to SAM-dependent methyltransferases, is necessary and sufficient to catalyze RQ biosynthesis from UQ in vitro. Remarkably, we show that RquA uses SAM as the amino group donor in a substitution reaction that converts UQ to RQ. In contrast to known aminotransferases, RquA does not use pyridoxal 5'-phosphate (PLP) as a coenzyme, but requires the presence of Mn2+ as a cofactor. As these findings reveal, RquA provides an example of a non-canonical SAM-dependent enzyme that does not catalyze methyl transfer, instead it uses SAM in an atypical amino transfer mechanism.