Trilok Neupane

Rhodoquinone (RQ) is a crucial electron carrier involved in anaerobic metabolism across select bacteria, protists, and animal species. Its biosynthesis is catalyzed by the rhodoquinone biosynthesis enzyme (RquA), a methyltransferase-like enzyme that uses S-adenosyl-L-methionine to transfer an amino group, converting ubiquinone (UQ) into RQ. The activity of RquA in vitro is enhanced by the presence of divalent metal cations. To probe the metal dependence of RquA, we characterized its interactions with Mn(II), Co(II), and Zn(II). We found that these metal cations bind to RquA with a 1:1 stoichiometry and that Mn(II) and Co(II) exhibited sub-micromolar binding affinities to RquA. Using Mn(II) as a spectroscopic probe, continuous‑wave electron paramagnetic resonance (EPR) revealed a single Mn(II) species with zero‑field splitting parameters |D| = 540(30) MHz and E/D = 0.30(3). Pulse EPR experiments on natural‑abundance and ¹⁵N‑labeled samples further identified a weakly‑to‑moderately coupled nitrogen ligand, with an isotropic hyperfine coupling constant |a_iso(¹⁵N)| = 2.7(1) MHz. Integrating these data with an AlphaFold3‑derived structural model, we propose a putative binding site for the catalytically required divalent metal cation, providing new insight into the structural basis of RquA function.

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.

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.

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-β-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 K_i 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.

RquA, a protein expressed from the rquA gene in bacteria and some protists, is required for the conversion of ubiquinone (UQ) to rhodoquinone (RQ). RQ is an essential electron carrier used in anaerobic metabolism. Sequence analysis of RquA has revealed an S ‐adenosyl methionine (SAM) binding domain. It has been hypothesized that SAM acts as the amino source or a catalyst for the conversion of UQ to RQ. The aim of this study was to determine the requirements for RquA function. Assays were performed in vitro under various conditions with refolded RquA and a synthetic UQ ₃ substrate. The resulting amounts of RQ ₃ produced were quantified using LC‐MS. In the absence of SAM, assays have not produced RQ ₃ , suggesting that SAM is required for RquA function. The requirement of zinc (II) has also been demonstrated, with assays performed in its absence failing to yield RQ ₃ . Increasing the concentration of either SAM or RquA present in the assay resulted in a corresponding increase in RQ ₃ . These results show that SAM and Zn ²⁺ are required for the function of RquA. Kinetic assays to further characterize RquA are currently underway.