Amidoximes can be used as prodrugs for amidines and related functional groups to enhance their intestinal absorption. These prodrugs are reduced to their active amidines. Other N-hydroxylated structures are mutagenic or responsible for toxic effects of drugs and are detoxified by reduction. In this study, a N-reductive enzyme system of pig liver mitochondria using benzamidoxime as a model substrate was identified. A protein fraction free from cytochrome b 5 and cytochrome b 5 reductase was purified, enhancing 250-fold the minor benzamidoxime-reductase activity catalyzed by the membrane-bound cytochrome b 5 /NADH cytochrome b 5 reductase system. This fraction contained a 35-kDa protein with homologies to the C-terminal domain of the human molybdenum cofactor sulfurase. Here it was demonstrated that this 35-kDa protein contains molybdenum cofactor and forms the hitherto ill defined third component of the N-reductive complex in the outer mitochondrial membrane. Thus, the 35-kDa protein represents a novel group of molybdenum proteins in eukaryotes as it forms the catalytic part of a three-component enzyme complex consisting of separate proteins. Supporting these findings, recombinant C-terminal domain of the human molybdenum cofactor sulfurase exhibited N-reductive activity in vitro, which was strictly dependent on molybdenum cofactor.

Background : SARS-CoV-2 seems to be mainly transmissible via respiratory droplets. We compared the time-dependent SARS-CoV-2 viral load in serial pharyngeal swab and exhaled breath (EB) samples of hospitalized COVID-19 patients. Methods : In this prospective proof of concept study, we examined hospitalized patients initially tested positive for SARS-CoV-2. The screening consisted of collecting paired oronasopharyngeal swab and EB specimens taken at different days of hospitalization. The EB collection was performed through a noninvasive simple method using an electret air filter-based device. SARS-CoV-2 RNA detection was determined with qRT-PCR. Results : Of 187 serial samples taken from 15 hospitalized patients, 87 oronasopharyngeal swabs and 70 of the 100 EB specimens tested positive. Comparing the number of SARS-CoV-2 copies, the viral load of the oronasopharyngeal swabs (n=87) was significantly higher (CI 99%, p<<0,001) than the viral load of the EB samples (n=70). The mean viral load per swab was 7.97 × 10 6 (1.65 × 10 2 -1.4 × 10 8 ), whereas EB samples showed 2.47 × 10 3 (7.19 × 10 1 -2.94 × 10 4 ) copies per 20 times exhaling. Viral loads of paired oronasopharyngeal swab and EB samples showed no correlation. Conclusions : Assessing the infectiousness of COVID-19 patients merely through pharyngeal swabs might not be accurate. Exhaled breath could represent a more suitable matrix for evaluating the infectiousness and might allow screening for superspreader individuals and widespread variants such as the Delta variant.

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.

Human molybdenum-containing enzyme mitochondrial amidoxime reducing component (mARC), cytochrome b 5 type B, and NADH cytochrome b 5 reductase form an N-reductive enzyme system that is capable of reducing N-hydroxylated compounds. Genetic variations are known, but their functional relevance is unclear. Our study aimed to investigate the incidence of nonsynonymous single nucleotide polymorphisms (SNPs) in the mARC genes in healthy Caucasian volunteers, to determine saturation of the protein variants with molybdenum cofactor (Moco), and to characterize the kinetic behavior of the protein variants by in vitro biotransformation studies. Genotype frequencies of six SNPs in the mARC genes (c.493A>G, c.560T>A, c.736T>A, and c.739G>C in MARC1; c.730G>A and c.735T>G in MARC2) were determined by pyrosequencing in a cohort of 340 healthy Caucasians. Protein variants were expressed in Escherichia coli. Saturation with Moco was determined by measurement of molybdenum by inductively coupled mass spectrometry. Steady state assays were performed with benzamidoxime. The six variants were of low frequency in this Caucasian population. Only one homozygous variant (c.493A; MARC1) was detected. All protein variants were able to bind Moco. Steady state assays showed statistically significant decreases of catalytic efficiency values for the mARC-2 wild type compared with the mARC-1 wild type (P < 0.05) and for two mARC-2 variants compared with the mARC-2 wild type (G244S, P < 0.05; C245W, P < 0.05). After simultaneous substitution of more than two amino acids in the mARC-1 protein, N-reductive activity was decreased 5-fold. One homozygous variant of MARC1 was detected in our sample. The encoded protein variant (A165T) showed no different kinetic parameters in the N-reduction of benzamidoxime.