Biotransformation enzymes ensure a viable homeostasis by regulating reversible cycles of oxidative and reductive reactions. The metabolism of nitrogen-containing compounds is of high pharmaceutical and toxicological relevance because N-oxygenated metabolites derived from reactions mediated by cytochrome P450 enzymes or flavin-dependent monooxygenases are in some cases highly toxic or mutagenic. The molybdenum-dependent mitochondrial amidoxime-reducing component (mARC) was found to be an extremely efficient counterpart, which is able to reduce the full range of N-oxygenated compounds and thereby mediates detoxification reactions. However, the 3D structure of this enzyme was unknown. Here we present the high-resolution crystal structure of human mARC. We give detailed insight into the coordination of its molybdenum cofactor (Moco), the catalytic mechanism, and its ability to reduce a wide range of N-oxygenated compounds. The identification of two key residues will allow future discrimination between mARC paralogs and ensure correct annotation. Since our structural findings contradict in silico predictions that are currently made by online databases, we propose domain definitions for members of the superfamily of Moco sulfurase C-terminal (MOSC) domain-containing proteins. Furthermore, we present evidence for an evolutionary role of mARC for the emergence of the xanthine oxidase protein superfamily. We anticipate the hereby presented crystal structure to be a starting point for future descriptions of MOSC proteins, which are currently poorly structurally characterized.

For the development of new drugs, the investigation of their metabolism is of central importance. In the past, the focus was mostly on the consideration of established enzymes leading to oxidations such as cytochrome P450. However, reductive metabolism by the mARC enzyme system can play an important role in particular for nitrogen containing functional groups. A rapid test was established to give developers of new drugs in the preclinical stage the opportunity to test the metabolism by mARC. To demonstrate the relevance and validity of the new test system, known and potential substrates were applied to this new assay. All known substrates could be detected by the system. Furthermore, several new substrates were found including long-established drugs such as hydroxyurea and new compounds in development such as epacdadostat.

The mitochondrial amidoxime reducing component is a recently discovered molybdenum enzyme in mammals which, in concert with the electron transport proteins cytochrome b5 and NADH cytochrome b5 reductase, catalyzes the reduction of -oxygenated structures. This three component enzyme system plays a major role in-reductive drug metabolism. Belonging to the group of -hydroxylated structures, hydroxamic acids are also potential substrates of the mARC-system. Hydroxamic acids show a variety of pharmacological activities and are therefore often found in drug candidates. They can also exhibit toxic properties as is the case for many aryl hydroxamic acids formed during the metabolism of arylamides. Biotransformation assays using recombinant human proteins, subcellular porcine tissue fractions as well as human cell culture were performed. Here the mARC-dependent reduction of the model compound benzhydroxamic acid is reported in addition to the reduction of three drugs. In comparison with other known substrates of the molybdenum depending enzyme system (e.g., amidoxime prodrugs) the conversion rates measured here are slower, thereby reflecting the mediocre metabolic stability and oral bioavailability of distinct hydroxamic acids. Moreover, the toxic-hydroxylated metabolite of the analgesic phenacetin, -hydroxyphenacetin, is not reduced by the mARC-system under the chosen conditions. This confirms the high toxicity of this component, as it needs to be detoxified by other pathways. This work highlights the need to monitor the-reductive metabolism of new drug candidates by the mARC-system when evaluating the metabolic stability of hydroxamic acid-containing structures or the potential risks of toxic metabolites.

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